Polypeptides Having Protease Activity

ABSTRACT

The present invention relates to isolated polypeptides having protease activity and isolated nucleic acid sequences encoding the proteases. The invention also relates to nucleic acid constructs, vectors, and host cells, including plant and animal cells, comprising the nucleic acid sequences, as well as methods for producing and using the proteases, in particular the use of the proteases in animal feed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 15/673,594filed on Aug. 10, 2017, now pending, which is a divisional of U.S.application Ser. No. 14/423,546 filed on Feb. 24, 2015, now U.S. Pat.No. 9,771,570, which is a 35 U.S.C. 371 national application ofinternational application no. PCT/EP2013/068361 filed on Sep. 5, 2013,which claims priority or the benefit under 35 U.S.C. 119 of Europeanapplication no. 12183079.8 filed on Sep. 5, 2012 and U.S. provisionalapplication No. 61/697,032 filed Sep. 5, 2012. The content of theseapplications is fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to isolated polypeptides having proteaseactivity and isolated nucleic acid sequences encoding the proteases. Theinvention also relates to nucleic acid constructs, vectors, and hostcells, including plant and animal cells, comprising the nucleic acidsequences, as well as methods for producing and using the proteases, inparticular, the use of the proteases in animal feed.

Background of the Invention

In the use of proteases in animal feed (in vivo), and/or the use of suchproteases for treating vegetable proteins (in vitro) it is noted thatproteins are essential nutritional factors for animals and humans.Humans and livestock usually get the necessary proteins from vegetableprotein sources. Important vegetable protein sources are, e.g., oilseedcrops, legumes and cereals.

When, e.g., soybean meal is included in the feed of mono-gastric animalssuch as pigs and poultry, a significant proportion of the soybean mealis not digested efficiently (the apparent ileal protein digestibility inpiglets, growing pigs and poultry such as broilers, laying hens androosters is only around 80%).

The gastrointestinal tract of animals consists of a series of segmentseach representing different pH environments. In mono-gastric animalssuch as pigs and poultry and many types of fish, the stomach is stronglyacidic with a pH potentially as low as 1-2, while the intestine has amore neutral pH of around 6-7.5. Apart from the stomach and intestine,poultry also have a crop preceding the stomach. The pH in the crop ismostly determined by the feed ingested and hence typically lies in therange of pH 4-6. Protein digestion by a protease may occur along theentire digestive tract, provided that the protease is active andsurvives the conditions in the digestive tract. Hence, proteases whichare highly acid stable and so can survive in the gastric environment andat the same time are efficiently active at the broad range ofphysiological pH of the digestive tract in the target animal areespecially desirable. The novel S53 proteases of the invention areuseful for these purposes.

Since animal feed is often formulated in pelleted form, in which steamis applied in the pelleting process, it is also desirable that proteasesused in animal feed are capable of remaining active after exposure tosaid steam treatment.

In order to produce a protease for industrial use, it is important thatthe protease is produced in high yields making the product available insufficient quantities in order to be able to provide the protease at afavourable price.

DESCRIPTION OF THE RELATED ART

S53 proteases are known in the art. An S53 peptide from Grifola frondosawith accession number MER078639 (SEQ ID NO: 9) has 83.6% sequenceidentity to SEQ ID NO: 5. An S53 protease from Postia placenta (UniProt:B8PMI5, SEQ ID NO: 10) was isolated by Martinez et al. having 74.5%sequence identity to SEQ ID NO: 5 in “Genome, transcriptome, andsecretome analysis of wood decay fungus Postia placenta supports uniquemechanisms of lignocellulose conversion”, 2009, Proc. Natl. Acad. Sci.USA 106:1954-1959.

Wymelenberg et al. have isolated an S53 protease (UniProt: Q281W2, SEQID NO: 11) in “Computational analysis of the Phanerochaete chrysosporiumv2.0 genome database and mass spectrometry identification of peptides inligninolytic cultures reveal complex mixtures of secreted proteins”,2006, Fungal Genet. Biol. 43:343-356 having 74.1% sequence identity toSEQ ID NO: 5. Another S53 polypeptide from Postia placenta(UniProt:B8P431, SEQ ID NO: 12) has been identified by Martinez et al.in “Genome, transcriptome, and secretome analysis of wood decay fungusPostia placenta supports unique mechanisms of lignocelluloseconversion”, 2009, Proc. Natl. Acad. Sci. U.S.A. 106:1954-1959 having68.2% sequence identity to SEQ ID NO: 5. Other peptides, including S53proteases, have less than 70% sequence identity to SEQ ID NO: 5.

Floudas et al. have published the sequence of an S53 protease in “ThePaleozoic origin of enzymatic lignin decomposition reconstructed from 31fungal genomes”, 2012, Science, 336:1715-1719 having 80.6% identity toSEQ ID NO: 5. Fernandez-Fueyo et al have published the sequences ofthree serine proteases in “Comparative genomics of Ceriporiopsissubvermispora and Phanerochaete chrysosporium provide insight intoselective ligninolysis”, 2012, Proc Natl Acad Sci USA. 109:5458-5463(UniProt:M2QQ01, SEQ ID NO: 26, UniProt:M2QWH2, SEQ ID NO: 27,UniProt:M2RD67, SEQ ID NO: 28) having 80.8%, 79.1% and 78.6% identity,respectively, to SEQ ID NO: 5.

WO 02/068623 describes a protease from Aspergillus niger with 49.2%sequence identity to SEQ ID NO: 5 for use in feed and food applications.WO 2012/048334 describes serine-type endopeptidases from Myceliophthorathermophila as a feed additive or for feedstuff with 47.9% sequenceidentity to SEQ ID NO: 5.

WO 95/28850 discloses the combination of a phytase and one or moremicrobial proteolytic enzymes to improve the solubility of vegetableproteins. WO 01/58275 discloses the use of acid stable proteases of thesubtilisin family in animal feed. WO 01/58276 discloses the use ofacid-stable proteases derived from Nocardiopsis sp. NRRL 18262 (the 10Rprotease), as well as a protease derived from Nocardiopsis alba DSM14010 in animal feed. WO 2004/072221, WO 2004/111220, WO 2004/111223, WO2005/035747, and WO 2005/123911 disclose proteases related to the 10Rprotease and their use in animal feed. WO 2004/072279 discloses the useof other proteases in animal feed. WO 2004/034776 discloses the use of asubtilisin/keratinase, PWD-1 from B. licheniformis, in the feed ofpoultry. WO 2004/077960 discloses a method for increasing thedigestibility of forage or grain in ruminants by applying a bacterial orfungal protease.

Commercial products comprising a protease and marketed for use in animalfeed include RONOZYME® ProAct (DSM NP/Novozymes), Axtra® (Danisco),Avizyme® (Danisco), Porzyme® (Danisco), Allzyme™ (Alltech), Versazyme®(BioResources, Int.), Poultrygrow™ (Jefo) and Cibenza® DP100 (Novus).

SUMMARY OF THE INVENTION

The present invention relates to isolated polypeptides having proteaseactivity selected from the group consisting of:

(a) a polypeptide having at least 84% sequence identity to thepolypeptide of SEQ ID NO: 5, SEQ ID NO: 6, or the mature polypeptide ofSEQ ID NO: 2 or SEQ ID NO: 4;

(b) a polypeptide having at least 83% sequence identity to thepolypeptide of SEQ ID NO: 19, or the mature polypeptide of SEQ ID NO: 16or SEQ ID NO: 18;

(c) a polypeptide having at least 85% sequence identity to thepolypeptide of SEQ ID NO: 24, or the mature polypeptide of SEQ ID NO: 21or SEQ ID NO: 23;

(d) a polypeptide encoded by a polynucleotide that hybridizes under highstringency conditions, or very high stringency conditions with

-   -   (i) the mature polypeptide coding sequence of SEQ ID NO: 1,    -   (ii) the mature polypeptide coding sequence of SEQ ID NO: 3,    -   (iii) the mature polypeptide coding sequence of SEQ ID NO: 15,    -   (iv) the mature polypeptide coding sequence of SEQ ID NO: 17,    -   (v) the mature polypeptide coding sequence of SEQ ID NO: 20,    -   (vi) the mature polypeptide coding sequence of SEQ ID NO: 22,    -   (vii) the full-length complementary strand of (i), (ii), (iii),        (iv), (v) or (vi);

(e) a polypeptide encoded by a polynucleotide having at least 84%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or SEQ ID NO: 3;

(f) a polypeptide encoded by a polynucleotide having at least 83%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 15 or SEQ ID NO: 17;

(g) a polypeptide encoded by a polynucleotide having at least 85%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 20 or SEQ ID NO: 22;

(h) a variant of the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 19 or SEQ ID NO: 24, or the mature polypeptide of SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 21 or SEQ ID NO: 23comprising a substitution, deletion, and/or insertion at one or more(several) positions; and

(i) a fragment of a polypeptide of (a), (b), (c), (d), (e), (f), (g) or(h) having protease activity.

The present invention also relates to the use of isolated polypeptidesin animal feed having protease activity selected from the groupconsisting of:

(a) a polypeptide having at least 60% sequence identity to thepolypeptide of SEQ ID NO: 5, SEQ ID NO: 6, or the mature polypeptide ofSEQ ID NO: 2 or SEQ ID NO: 4;

(b) a polypeptide having at least 60% sequence identity to thepolypeptide of SEQ ID NO: 19, or the mature polypeptide of SEQ ID NO: 16or SEQ ID NO: 18;

(c) a polypeptide having at least 60% sequence identity to thepolypeptide of SEQ ID NO: 24, or the mature polypeptide of SEQ ID NO: 21or SEQ ID NO: 23;

(d) a polypeptide encoded by a polynucleotide that hybridizes under highstringency conditions, or very high stringency conditions with

-   -   (i) the mature polypeptide coding sequence of SEQ ID NO: 1,    -   (ii) the mature polypeptide coding sequence of SEQ ID NO: 3,    -   (iii) the mature polypeptide coding sequence of SEQ ID NO: 15,    -   (iv) the mature polypeptide coding sequence of SEQ ID NO: 17,    -   (v) the mature polypeptide coding sequence of SEQ ID NO: 20,    -   (vi) the mature polypeptide coding sequence of SEQ ID NO: 22,    -   (vii) the full-length complementary strand of (i), (ii), (iii),        (iv), (v) or (vi);

(e) a polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or SEQ ID NO: 3;

(f) a polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 15 or SEQ ID NO: 17;

(g) a polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 20 or SEQ ID NO: 22;

(h) a variant of the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 19 or SEQ ID NO: 24, or the mature polypeptide of SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 21 or SEQ ID NO: 23comprising a substitution, deletion, and/or insertion at one or more(several) positions; and

(i) a fragment of a polypeptide of (a), (b), (c), (d), (e), (f), (g) or(h) having protease activity.

The present invention relates to isolated polynucleotides encoding thepolypeptides of the present invention, nucleic acid constructs,recombinant expression vectors, and recombinant host cells comprisingthe polynucleotides, and to methods of producing the polypeptides.

The present invention also relates to compositions, preferably animalfeed compositions, comprising the polypeptides of the invention; use ofthe polypeptides of the invention in animal feed or as animal feedadditives; methods for preparing a composition for use in animal feed,for improving the nutritional value of an animal feed, and methods oftreating proteins to be used in animal feed compositions.

Overview of Sequence Listing

SEQ ID NO: 1 is the cDNA sequence of S53 protease 3 as isolated fromMeripilus giganteus.

SEQ ID NO: 2 is the amino acid sequence as deduced from SEQ ID NO: 1.

SEQ ID NO: 3 is the DNA sequence of the recombinant expressed DNAsequence from SEQ ID NO: 1 with HQ-tag.

SEQ ID NO: 4 is the amino acid sequence as deduced from SEQ ID NO: 3.

SEQ ID NO: 5 is the amino acid sequence of the mature S53 protease 3from Meripilus giganteus.

SEQ ID NO: 6 is the amino acid sequence of the mature S53 proteaseobtained from SEQ ID NO. 3.

SEQ ID NO: 7 is the DNA sequence of protease 10R (WO 2005/035747, SEQ IDNO: 1).

SEQ ID NO: 8 is the amino acid sequence of protease 10R (WO 2005/035747,SEQ ID NO: 2).

SEQ ID NO: 9 is the amino acid sequence of an S53 peptide from Grifolafrondosa (MER078639).

SEQ ID NO: 10 is the amino acid sequence of an S53 peptide from Postiaplacenta (UniProt: B8PMI5).

SEQ ID NO: 11 is the amino acid sequence of an S53 peptide fromPhanerochaete chrysosporium (UniProt: Q281W2).

SEQ ID NO: 12 is the amino acid sequence of an S53 peptide from Postiaplacenta (UniProt: B8P431).

SEQ ID NO: 13 is primer 597.

SEQ ID NO: 14 is primer 598.

SEQ ID NO: 15 is the cDNA sequence of S53 protease 1 isolated fromTrametes cf. versicolor.

SEQ ID NO: 16 is the amino acid sequence as deduced from SEQ ID NO: 15.

SEQ ID NO: 17 is the DNA sequence of the recombinant expressed DNAsequence from SEQ ID NO: 15.

SEQ ID NO: 18 is the amino acid sequence as deduced from SEQ ID NO: 17.

SEQ ID NO: 19 is the amino acid sequence of the mature S53 proteaseobtained from SEQ ID NO. 15 and SEQ ID NO: 17.

SEQ ID NO: 20 is the cDNA sequence of S53 protease 2 isolated fromTrametes versicolor.

SEQ ID NO: 21 is the amino acid sequence as deduced from SEQ ID NO: 20.

SEQ ID NO: 22 is the DNA sequence of the recombinant expressed DNAsequence from SEQ ID NO: 20.

SEQ ID NO: 23 is the amino acid sequence as deduced from SEQ ID NO: 22.

SEQ ID NO: 24 is the amino acid sequence of the mature S53 proteaseobtained from SEQ ID NO. 20 and SEQ ID NO: 22.

SEQ ID NO: 25 is the amino acid sequence of an S53 peptide fromDichomitus squalens (UniProt: R7SPH9).

SEQ ID NO: 26 is the amino acid sequence of an S53 peptide fromCeriporiopsis subvermispora (UniProt: M2QQ01).

SEQ ID NO: 27 is the amino acid sequence of an S53 peptide fromCeriporiopsis subvermispora (UniProt: M2QWH2).

SEQ ID NO: 28 is the amino acid sequence of an S53 peptide fromCeriporiopsis subvermispora (UniProt: M2RD67).

Identity Matrix of sequences: SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQ SEQSEQ ID: 2 ID: 5 ID: 9 ID: 18 ID: 19 ID: 23 ID: 24 ID: 25 ID: 26 ID: 27ID: 28 SEQ 100 100 79.8 86.7 86.6 86.0 85.5 76.2 75.0 73.1 72.8 ID: 2SEQ 100 100 83.6 86.6 86.6 85.5 85.5 80.6 80.8 79.1 78.6 ID: 5 SEQ 79.883.6 100 79.6 84.1 78.6 82.7 72.7 78.0 75.8 76.4 ID: 9 SEQ 86.7 86.679.6 100 100 96.5 96.2 77.8 77.0 75.0 74.8 ID: 18 SEQ 86.6 86.6 84.1 100100 96.2 96.2 81.4 82.5 79.4 79.7 ID: 19 SEQ 86.0 85.5 78.6 96.5 96.2100 100 76.8 77.1 75.0 74.7 ID: 23 SEQ 85.5 85.5 82.7 96.2 96.2 100 10080.0 82.2 79.1 79.5 ID: 24 SEQ 76.2 80.6 72.7 77.8 81.4 76.8 80.0 10070.4 68.9 69.4 ID: 25 SEQ 75.0 80.8 78.0 77.0 82.5 77.1 82.2 70.4 10093.0 94.2 ID: 26 SEQ 73.1 79.1 75.8 75.0 79.4 75.0 79.1 68.9 93.0 10094.4 ID: 27 SEQ 72.8 78.6 76.4 74.8 79.7 74.7 79.5 69.4 94.2 94.4 100ID: 28

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the pH-activity profile of the S53 protease 3 fromMeripilus giganteus (SEQ ID NO: 3) (from example 2) compared to protease10R on the Suc-AAPF-pNA substrate at 25° C.

FIG. 2 shows the pH-stability profile of the S53 protease 3 fromMeripilus giganteus (SEQ ID NO: 3) (from example 2) compared to protease10R (residual activity after 2 hours at 37° C.).

FIG. 3 shows the temperature activity profile of the S53 protease 3 fromMeripilus giganteus (SEQ ID NO: 3) (from example 2) at pH 4.0 comparedto protease 10R on Protazyme AK at pH 6.5.

FIG. 4 shows the P1-specificity of the S53 protease 3 from Meripilusgiganteus (SEQ ID NO: 3) (from example 2) at pH 4 compared to protease10R at pH 9.0 on 10 Suc-AAPX-pNA substrates, 25° C.

FIG. 5 shows the activity (OD₃₄₀× dilution factor) on soybean-maize mealof the S53 protease 3 from Meripilus giganteus (SEQ ID NO: 3) (fromexample 2) compared to protease 10R.

FIG. 6 shows the level of free amines (OD₃₄₀× dilution factor) in BlankTo samples, Blank samples and samples incubated with the S53 protease 3from Meripilus giganteus (SEQ ID NO: 3) (from example 2) or protease10R.

FIG. 7 shows the pH-activity profile of the S53 protease 1 isolated fromTrametes cf. versicolor (SEQ ID NO: 16) compared to the S53 protease 3from Meripilus giganteus (SEQ ID NO: 3) (from example 2) on theSuc-AAPF-pNA substrate at 25° C.

FIG. 8 shows the pH-stability profile of the S53 protease 1 isolatedfrom Trametes cf. versicolor (SEQ ID NO: 16) compared to the S53protease 3 from Meripilus giganteus (SEQ ID NO: 3) (from example 2)(residual activity after 2 hours at 37° C.).

FIG. 9 shows the temperature activity profile of the S53 protease 1isolated from Trametes cf. versicolor SEQ ID NO: 16) compared to the S53protease 3 from Meripilus giganteus (SEQ ID NO: 3) (from example 2) onProtazyme AK at pH 4.

FIG. 10 shows the P1-specificity of the S53 protease 1 isolated fromTrametes cf. versicolor SEQ ID NO: 16) compared to the S53 protease 3from Meripilus giganteus (SEQ ID NO: 3) (from example 2) at pH 4 on 10Suc-AAPX-pNA substrates, 25° C.

DEFINITIONS

Allelic variant: The term “allelic variant” means any of two or morealternative forms of a gene occupying the same chromosomal locus.Allelic variation arises naturally through mutation, and may result inpolymorphism within populations. Gene mutations can be silent (no changein the encoded polypeptide) or may encode polypeptides having alteredamino acid sequences. An allelic variant of a polypeptide is apolypeptide encoded by an allelic variant of a gene.

cDNA: The term “cDNA” means a DNA molecule that can be prepared byreverse transcription from a mature, spliced, mRNA molecule obtainedfrom a eukaryotic cell. cDNA lacks intron sequences that may be presentin the corresponding genomic DNA. The initial, primary RNA transcript isa precursor to mRNA that is processed through a series of steps,including splicing, before appearing as mature spliced mRNA.

Coding sequence: The term “coding sequence” means a polynucleotide,which directly specifies the amino acid sequence of a polypeptide. Theboundaries of the coding sequence are generally determined by an openreading frame, which usually begins with the ATG start codon oralternative start codons such as GTG and TTG and ends with a stop codonsuch as TAA, TAG, and TGA. The coding sequence may be a DNA, cDNA,synthetic, or recombinant polynucleotide.

Control sequences: The term “control sequences” means nucleic acidsequences necessary for expression of a polynucleotide encoding a maturepolypeptide of the present invention. Each control sequence may benative (i.e., from the same gene) or foreign (i.e., from a differentgene) to the polynucleotide encoding the polypeptide or native orforeign to each other.

Such control sequences include, but are not limited to, a leader,polyadenylation sequence, propeptide sequence, promoter, signal peptidesequence, and transcription terminator. At a minimum, the controlsequences include a promoter, and transcriptional and translational stopsignals. The control sequences may be provided with linkers for thepurpose of introducing specific restriction sites facilitating ligationof the control sequences with the coding region of the polynucleotideencoding a polypeptide.

Expression: The term “expression” includes any step involved in theproduction of the polypeptide including, but not limited to,transcription, post-transcriptional modification, translation,post-translational modification, and secretion.

Expression vector: The term “expression vector” means a linear orcircular DNA molecule that comprises a polynucleotide encoding apolypeptide and is operably linked to additional nucleotides thatprovide for its expression.

Fragment: The term “fragment” means a polypeptide having one or more(e.g., several) amino acids deleted from the amino and/or carboxylterminus of a mature polypeptide; wherein the fragment has proteaseactivity. In one aspect, a fragment contains at least 330 amino acidresidues (e.g., amino acids 20 to 349 of SEQ ID NO: 2 or SEQ ID NO: 5);in another aspect, a fragment contains at least 345 amino acid residues(e.g., amino acids 10 to 354 of SEQ ID NO: 2 or SEQ ID NO: 5); in afurther aspect, a fragment contains at least 355 amino acid residues(e.g., amino acids 5 to 359 of SEQ ID NO: 2 or SEQ ID NO: 5). In oneaspect, a fragment contains at least 330 amino acid residues (e.g.,amino acids 20 to 349 of SEQ ID NO: 16 or SEQ ID NO: 20); in anotheraspect, a fragment contains at least 345 amino acid residues (e.g.,amino acids 10 to 354 of SEQ ID NO: 16 or SEQ ID NO: 20); in a furtheraspect, a fragment contains at least 355 amino acid residues (e.g.,amino acids 5 to 359 of SEQ ID NO: 16 or SEQ ID NO: 20). In one aspect,a fragment contains at least 330 amino acid residues (e.g., amino acids20 to 349 of SEQ ID NO: 21 or SEQ ID NO: 24); in another aspect, afragment contains at least 345 amino acid residues (e.g., amino acids 10to 354 of SEQ ID NO: 21 or SEQ ID NO: 24); in a further aspect, afragment contains at least 355 amino acid residues (e.g., amino acids 5to 359 of SEQ ID NO: 21 or SEQ ID NO: 24).

Host cell: The term “host cell” means any cell type that is susceptibleto transformation, transfection, transduction, and the like with anucleic acid construct or expression vector comprising a polynucleotideof the present invention. The term “host cell” encompasses any progenyof a parent cell that is not identical to the parent cell due tomutations that occur during replication.

Isolated polynucleotide: The term “isolated polynucleotide” means apolynucleotide that is in a form or environment that does not occur innature, such as (1) any non-naturally occurring polynucleotide, (2) anypolynucleotide that is at least partially removed from one or more orall of the naturally occurring constituents with which it is associatedin nature; (3) any polynucleotide that is modified by the hand of manrelative to that polynucleotide as found in nature or (4) anypolynucleotide modified by increasing the amount of the polynucleotiderelative to other components with which it is naturally associated(e.g., recombinant production in a host cell; multiple copies of a geneencoding the substance; and use of a stronger promoter than the promoternaturally associated with the gene encoding the substance). In oneaspect, the isolated polynucleotide is at least 1% pure, e.g., at least5% pure, more at least 10% pure, at least 20% pure, at least 40% pure,at least 60% pure, at least 80% pure, at least 90% pure, and at least95% pure, as determined by agarose electrophoresis. The polynucleotidesmay be of genomic, cDNA, RNA, semisynthetic, synthetic origin, or anycombinations thereof.

Isolated polypeptide: The term “isolated polypeptide” means apolypeptide that is in a form or environment that does not occur innature, such as (1) any non-naturally occurring polypeptide, (2) anypolypeptide that is at least partially removed from one or more or allof the naturally occurring constituents with which it is associated innature; (3) any polypeptide that is modified by the hand of man relativeto that polypeptide as found in nature in admixture with othercomponents, such as other polypeptides, secondary metabolites, salts, etalia or (4) any polypeptide modified by increasing the amount of thepolypeptide relative to other components with which it is naturallyassociated. In one aspect, the polypeptide is at least 1% pure, e.g., atleast 5% pure, at least 10% pure, at least 20% pure, at least 40% pure,at least 60% pure, at least 80% pure, and at least 90% pure, asdetermined by SDS-PAGE.

Mature polypeptide: The term “mature polypeptide” means a polypeptide inits final form following translation and any post-translationalmodifications, such as N-terminal processing, C-terminal truncation,glycosylation, phosphorylation, etc. In one aspect, the maturepolypeptide is amino acids 1 to 366 in the numbering of SEQ ID NO: 2based on sequencing using Edman degradation and intact molecular weightanalysis of the mature polypeptide with C-terminal HQ-tag. Using theprediction program SignalP (Nielsen et al., 1997, Protein Engineering10: 1-6), amino acids −198 to −182 in the numbering of SEQ ID NO: 2 arepredicted to be the signal peptide.

In another aspect, the mature polypeptide is amino acids 1 to 366 in thenumbering of SEQ ID NO: 17 based on sequencing using Edman degradationand intact molecular weight analysis of the mature polypeptide. Usingthe prediction program SignalP (Nielsen et al., 1997, ProteinEngineering 10: 1-6), amino acids −199 to −183 in the numbering of SEQID NO: 17 are predicted to be the signal peptide.

In a further aspect, the mature polypeptide in the numbering of SEQ IDNO: 23 is predicted to be amino acids 1 to 366 and the signal peptide ispredicted to be amino acids −199 to −183 based on the prediction programSignalP (Nielsen et al., 1997, Protein Engineering 10: 1-6). It is knownin the art that a host cell may produce a mixture of two of moredifferent mature polypeptides (i.e., with a different C-terminal and/orN-terminal amino acid) expressed by the same polynucleotide.

Mature polypeptide coding sequence: The term “mature polypeptide codingsequence” means a polynucleotide that encodes a mature polypeptidehaving protease activity. In one aspect, the mature polypeptide codingsequence is nucleotides 605 to 1702 in the numbering of SEQ ID NO: 1based on the determination of the mature polypeptide by Edmandegradation and intact molecular weight analysis of the maturepolypeptide with C-terminal HQ-tag. Furthermore, nucleotides 11 to 61 inthe numbering of SEQ ID NO: 1 are predicted to encode a signal peptidebased on the prediction program SignalP (Nielsen et al., 1997, ProteinEngineering 10: 1-6).

In another aspect, the mature polypeptide coding sequence is the joinedsequence of nucleotides 707 to 853, nucleotides 912 to 1022, nucleotides1077 to 1276, nucleotides 1332 to 1469, nucleotides 1531 to 1978 andnucleotides 2031 to 2084 of SEQ ID NO: 15 or the cDNA sequence thereofbased on the determination of the mature polypeptide by Edmandegradation and intact molecular weight analysis of the maturepolypeptide. Nucleotides 1 to 51 in the numbering of SEQ ID NO: 15 arepredicted to encode a signal peptide based on the prediction programSignalP (Nielsen et al., 1997, Protein Engineering 10: 1-6). In afurther aspect, the mature polypeptide coding sequence is nucleotides598 to 1695 of SEQ ID NO: 17 or the cDNA sequence thereof based on thedetermination of the mature polypeptide by Edman degradation and intactmolecular weight analysis of the mature polypeptide. Nucleotides 1 to 51in the numbering of SEQ ID NO: 22 are predicted to encode a signalpeptide based on the prediction program SignalP (Nielsen et al., 1997,Protein Engineering 10: 1-6).

In another aspect, the mature polypeptide coding sequence is predictedto be the joined sequence of nucleotides 706 to 852, nucleotides 914 to1024, nucleotides 1080 to 1279, nucleotides 1333 to 1470, nucleotides1532 to 1979 and nucleotides 2032 to 2085 of SEQ ID NO: 20 or the cDNAsequence thereof based on the SignalP program (Nielsen et al., 1997,supra) that predicts nucleotides 1 to 51 of SEQ ID NO: 20 encode asignal peptide. In another aspect, the mature polypeptide codingsequence is predicted to be nucleotides 598 to 1695 of SEQ ID NO: 22 orthe cDNA sequence thereof based on the SignalP program (Nielsen et al.,1997, supra) that predicts nucleotides 1 to 51 of SEQ ID NO: 22 encode asignal peptide.

Nucleic acid construct: The term “nucleic acid construct” means anucleic acid molecule, either single- or double-stranded, which isisolated from a naturally occurring gene or is modified to containsegments of nucleic acids in a manner that would not otherwise exist innature or which is synthetic. The term nucleic acid construct issynonymous with the term “expression cassette” when the nucleic acidconstruct contains the control sequences required for expression of acoding sequence of the present invention.

Operably linked: The term “operably linked” means a configuration inwhich a control sequence is placed at an appropriate position relativeto the coding sequence of a polynucleotide such that the controlsequence directs the expression of the coding sequence.

Protease activity: The term “protease activity” means proteolyticactivity (EC 3.4). There are several protease activity types such astrypsin-like proteases cleaving at the carboxy-terminal side of Arg andLys residues and chymotrypsin-like proteases cleaving at thecarboxy-terminal side of hydrophobic amino acid residues. Proteases ofthe invention are serine endopeptidases (EC 3.4.21) with acidicpH-optimum (pH optimum <pH 7).

Protease activity can be measured using any assay, in which a substrateis employed, that includes peptide bonds relevant for the specificity ofthe protease in question. Assay-pH and assay-temperature are likewise tobe adapted to the protease in question. Examples of assay-pH-values arepH 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Examples of assay-temperaturesare 15, 20, 25, 30, 35, 37, 40, 45, 50, 55, 60, 65, 70, 80, 90, or 95°C. Examples of general protease substrates are casein, bovine serumalbumin and haemoglobin. In the classical Anson and Mirsky method,denatured haemoglobin is used as substrate and after the assayincubation with the protease in question, the amount of trichloroaceticacid soluble haemoglobin is determined as a measurement of proteaseactivity (Anson and Mirsky, 1932, J. Gen. Physiol. 16: 59 and Anson,1938, J. Gen. Physiol. 22: 79).

For the purpose of the present invention, protease activity wasdetermined using assays which are described in “Materials and Methods”,such as the Kinetic Suc-AAPF-pNA assay, Protazyme AK assay, KineticSuc-AAPX-pNA assay and o-Phthaldialdehyde (OPA). For the Protazyme AKassay, insoluble Protazyme AK (Azurine-Crosslinked Casein) substrateliberates a blue colour when incubated with the protease and the colouris determined as a measurement of protease activity. For theSuc-AAPF-pNA assay, the colorless Suc-AAPF-pNA substrate liberatesyellow paranitroaniline when incubated with the protease and the yellowcolor is determined as a measurement of protease activity.

The polypeptides of the present invention have at least 20%, e.g., atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, and at least 100% of the protease activity ofthe polypeptide of SEQ ID NO: 6, SEQ ID NO: 19 and/or SEQ D NO: 24.

Sequence Identity: The relatedness between two amino acid sequences orbetween two nucleotide sequences is described by the parameter “sequenceidentity”.

For purposes of the present invention, the degree of sequence identitybetween two amino acid sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol.48: 443-453) as implemented in the Needle program of the EMBOSS package(EMBOSS: The European Molecular Biology Open Software Suite, Rice etal., 2000, Trends Genet. 16: 276-277), preferably version 3.0.0 orlater. Version 6.1.0 was used. The optional parameters used are gap openpenalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSSversion of BLOSUM62) substitution matrix. The output of Needle labeled“longest identity” (obtained using the -nobrief option) is used as thepercent identity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of sequence identitybetween two deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,supra), preferably version 3.0.0 or later. Version 6.1.0 was used. Theoptional parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4)substitution matrix. The output of Needle labeled “longest identity”(obtained using the -nobrief option) is used as the percent identity andis calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment)

Stringency conditions: The different strigency conditions are defined asfollows.

The term “very low stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 25% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS at 45° C.

The term “low stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 25% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 50° C.

The term “medium stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 35% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 55° C.

The term “medium-high stringency conditions” means for probes of atleast 100 nucleotides in length, prehybridization and hybridization at42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and 35% formamide, following standard Southernblotting procedures for 12 to 24 hours. The carrier material is finallywashed three times each for 15 minutes using 2×SSC, 0.2% SDS at 60° C.

The term “high stringency conditions” means for probes of at least 100nucleotides in length, prehybridization and hybridization at 42° C. in5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon spermDNA, and 50% formamide, following standard Southern blotting proceduresfor 12 to 24 hours. The carrier material is finally washed three timeseach for 15 minutes using 2×SSC, 0.2% SDS at 65° C.

The term “very high stringency conditions” means for probes of at least100 nucleotides in length, prehybridization and hybridization at 42° C.in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmonsperm DNA, and 50% formamide, following standard Southern blottingprocedures for 12 to 24 hours. The carrier material is finally washedthree times each for 15 minutes using 2×SSC, 0.2% SDS at 70° C.

Subsequence: The term “subsequence” means a polynucleotide having one ormore (several) nucleotides deleted from the 5′ and/or 3′ end of a maturepolypeptide coding sequence; wherein the subsequence encodes a fragmenthaving protease activity. In one aspect, a subsequence contains at least990 nucleotides (e.g., nucleotides 662 to 1651 of SEQ ID NO: 1), e.g.,at least 1035 nucleotides (e.g., nucleotides 632 to 1666 of SEQ ID NO:1); e.g., at least 1065 nucleotides (e.g., nucleotides 617 to 1681 ofSEQ ID NO: 1).

Substantially pure polynucleotide: The term “substantially purepolynucleotide” means a polynucleotide preparation free of otherextraneous or unwanted nucleotides and in a form suitable for use withingenetically engineered polypeptide production systems. Thus, asubstantially pure polynucleotide contains at most 10%, at most 8%, atmost 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%, andat most 0.5% by weight of other polynucleotide material with which it isnatively or recombinantly associated. A substantially purepolynucleotide may, however, include naturally occurring 5′ and 3′untranslated regions, such as promoters and terminators. Preferably, thepolynucleotide is at least 90% pure, e.g., at least 92% pure, at least94% pure, at least 95% pure, at least 96% pure, at least 97% pure, atleast 98% pure, at least 99% pure, and at least 99.5% pure or 100% pureby weight. The polynucleotides of the present invention are preferablyin a substantially pure form.

Substantially pure polypeptide: The term “substantially purepolypeptide” means a preparation that contains at most 10%, at most 8%,at most 6%, at most 5%, at most 4%, at most 3%, at most 2%, at most 1%,and at most 0.5% by weight of other polypeptide material with which itis natively or recombinantly associated. Preferably, the polypeptide isat least 92% pure, e.g., at least 94% pure, at least 95% pure, at least96% pure, at least 97% pure, at least 98% pure, at least 99%, at least99.5% pure, and 100% pure by weight of the total polypeptide materialpresent in the preparation. The polypeptides of the present inventionare preferably in a substantially pure form. This can be accomplished,for example, by preparing the polypeptide by well-known recombinantmethods or by classical purification methods.

Variant: The term “variant” means a polypeptide having protease activitycomprising an alteration, i.e., a substitution, insertion, and/ordeletion of one or more (several) amino acid residues at one or more(several) positions. A substitution means a replacement of an amino acidoccupying a position with a different amino acid; a deletion meansremoval of an amino acid occupying a position; and an insertion meansadding 1-3 amino acids adjacent to an amino acid occupying a position.The variants of the present invention have at least 20%, e.g., at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, at least 95%, or at least 100% of the protease activity of thepolypeptide of SEQ ID NO: 5, SEQ ID NO: 6, or the mature polypeptide ofSEQ ID NO: 2 or SEQ ID NO: 4.

DETAILED DESCRIPTION OF THE INVENTION Polypeptides Having ProteaseActivity

Polypeptides having protease activity, or proteases, are sometimes alsodesignated peptidases, proteinases, peptide hydrolases, or proteolyticenzymes. Proteases may be of the exo-type that hydrolyse peptidesstarting at either end thereof, or of the endo-type that act internallyin polypeptide chains (endopeptidases). Endopeptidases show activity onN- and C-terminally blocked peptide substrates that are relevant for thespecificity of the protease in question.

The term “protease” is defined herein as an enzyme that hydrolysespeptide bonds. This definition of protease also applies to theprotease-part of the terms “parent protease” and “protease variant,” asused herein. The term “protease” includes any enzyme belonging to the EC3.4 enzyme group (including each of the eighteen subclasses thereof).The EC number refers to Enzyme Nomenclature 1992 from NC-IUBMB, AcademicPress, San Diego, Calif., including supplements 1-5 published in 1994,Eur. J. Biochem. 223: 1-5; 1995, Eur. J. Biochem. 232: 1-6; 1996, Eur.J. Biochem. 237: 1-5; 1997, Eur. J. Biochem. 250: 1-6; and 1999, Eur. J.Biochem. 264: 610-650 respectively. The nomenclature is regularlysupplemented and updated; see, e.g., the World Wide Web (WWW) atwww.chem.qmw.ac.uk/iubmb/enzyme/index.html.

The proteases of the invention and for use according to the inventionare selected from the group consisting of:

(a) proteases belonging to the EC 3.4.21. enzyme group; and/or

(b) proteases belonging to the EC 3.4.14. enzyme group; and/or

(c) Serine proteases of the peptidase family S53 that comprises twodifferent types of peptidases: tripeptidyl aminopeptidases (exo-type)and endo-peptidases; as described in 1993, Biochem. J. 290:205-218 andin MEROPS protease database, release 9.4 (31 Jan. 2011)(www.merops.ac.uk). The database is described in Rawlings et al., 2010,“MEROPS: the peptidase database”, Nucl. Acids Res. 38: D227-D233.

For determining whether a given protease is a Serine protease, and afamily S53 pro-tease, reference is made to the above Handbook and theprinciples indicated therein. Such determination can be carried out forall types of proteases, be it naturally occurring or wild-typeproteases; or genetically engineered or synthetic proteases.

Peptidase family S53 contains acid-acting endopeptidases andtripeptidyl-peptidases. The residues of the catalytic triad are Glu,Asp, Ser, and there is an additional acidic residue, Asp, in theoxyanion hole. The order of the residues is Glu, Asp, Asp, Ser. The Serresidue is the nucleophile equivalent to Ser in the Asp, His, Ser triadof subtilisin, and the Glu of the triad is a substitute for the generalbase, His, in subtilisin.

Mutation of any of the amino acids of the catalytic triad or oxyanionhole will result in a change or loss of enzyme activity. The amino acidsof the catalytic triad and oxyanion hole of the S53 protease 3 fromMeripilus giganteus (SEQ ID NO: 5) are probably positions Glu-85,Asp-89, Asp-175 and Ser-283. The amino acids of the catalytic triad andoxyanion hole of the S53 protease 1 from Trametes versicolor (SEQ ID NO:19) are probably positions Glu-85, Asp-89, Asp-175 and Ser-283. Theamino acids of the catalytic triad and oxyanion hole of the S53 protease2 from Trametes versicolor (SEQ ID NO: 24) are probably positionsGlu-85, Asp-89, Asp-175 and Ser-283.

The peptidases of the S53 family tend to be most active at acidic pH(unlike the homologous subtilisins), and this can be attributed to thefunctional importance of carboxylic residues, notably Asp in theoxyanion hole. The amino acid sequences are not closely similar to thosein family S8 (i.e., serine endopeptidase subtilisins and homologues),and this, taken together with the quite different active site residuesand the resulting lower pH for maximal activity, provides for asubstantial difference to that family. Protein folding of the peptidaseunit for members of this family resembles that of subtilisin, having theclan type SB.

A new S53 protease from Meripilus giganteus with high activity at low pH(3-4) on soybean-maize meal was identified and cloned in relation to thepresent invention. For determining whether a given protease is a Serineprotease, and a family S53 protease, reference is made to the aboveHandbook and the principles indicated therein. Such determination can becarried out for all types of proteases, be it naturally occurring orwild-type proteases; or genetically engineered or synthetic proteases.

The present invention provides polypeptides having protease activity andpolynucleotides encoding the polypeptides. The proteases of theinvention are serine proteases of the peptidase family S53. Theproteases of the invention exhibit pH properties, especially pHstability properties, which make them of substantial interest ascandidates for use in animal feed, and other applications.

The proteases of the invention are acidic proteases with a preferencefor hydrophibic amino acid residues such as Leu, Tyr, Phe and Met in theP1 position. The proteases have high activity on Suc-Ala-Ala-Pro-Leu-pNAand Suc-Ala-Ala-Pro-Phe-pNA with a broad pH range from 2-5 and retainmore than 95% activity after being subjected for 2 hours to pH as low as3.

The present invention relates to isolated polypeptides having proteaseactivity selected from the group consisting of:

(a) a polypeptide having at least 84% sequence identity to thepolypeptide of SEQ ID NO: 5, SEQ ID NO: 6, or the mature polypeptide ofSEQ ID NO: 2 or SEQ ID NO: 4;

(b) a polypeptide having at least 83% sequence identity to thepolypeptide of SEQ ID NO: 19, or the mature polypeptide of SEQ ID NO: 16or SEQ ID NO: 18;

(c) a polypeptide having at least 85% sequence identity to thepolypeptide of SEQ ID NO: 24, or the mature polypeptide of SEQ ID NO: 21or SEQ ID NO: 23;

(d) a polypeptide encoded by a polynucleotide that hybridizes under highstringency conditions, or very high stringency conditions with

-   -   (i) the mature polypeptide coding sequence of SEQ ID NO: 1,    -   (ii) the mature polypeptide coding sequence of SEQ ID NO: 3,    -   (iii) the mature polypeptide coding sequence of SEQ ID NO: 15,    -   (iv) the mature polypeptide coding sequence of SEQ ID NO: 17,    -   (v) the mature polypeptide coding sequence of SEQ ID NO: 20,    -   (vi) the mature polypeptide coding sequence of SEQ ID NO: 22,    -   (vii) the full-length complementary strand of (i), (ii), (iii),        (iv), (v) or (vi);

(e) a polypeptide encoded by a polynucleotide having at least 84%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or SEQ ID NO: 3;

(f) a polypeptide encoded by a polynucleotide having at least 83%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 15 or SEQ ID NO: 17;

(g) a polypeptide encoded by a polynucleotide having at least 85%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 20 or SEQ ID NO: 22;

(h) a variant of the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 19 or SEQ ID NO: 24, or the mature polypeptide of SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 21 or SEQ ID NO: 23comprising a substitution, deletion, and/or insertion at one or more(several) positions; and

(i) a fragment of a polypeptide of (a), (b), (c), (d), (e), (f), (g) or(h) having protease activity.

An embodiment of the invention is a polypeptide having at least 85%sequence identity to the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, orthe mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

An embodiment of the invention is a polypeptide having at least 86%sequence identity to the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, orthe mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

An embodiment of the invention is a polypeptide having at least 87%sequence identity to the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, orthe mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

An embodiment of the invention is a polypeptide having at least 88%sequence identity to the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, orthe mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

An embodiment of the invention is a polypeptide having at least 89%sequence identity to the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, orthe mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

An embodiment of the invention is a polypeptide having at least 90%sequence identity to the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, orthe mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

An embodiment of the invention is a polypeptide having at least 91%sequence identity to the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, orthe mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

An embodiment of the invention is a polypeptide having at least 92%sequence identity to the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, orthe mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

An embodiment of the invention is a polypeptide having at least 93%sequence identity to the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, orthe mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

An embodiment of the invention is a polypeptide having at least 94%sequence identity to the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, orthe mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

An embodiment of the invention is a polypeptide having at least 95%sequence identity to the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, orthe mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

An embodiment of the invention is a polypeptide having at least 96%sequence identity to the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, orthe mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

An embodiment of the invention is a polypeptide having at least 97%sequence identity to the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, orthe mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

An embodiment of the invention is a polypeptide having at least 98%sequence identity to the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, orthe mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

An embodiment of the invention is a polypeptide having at least 99%sequence identity to the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, orthe mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

An embodiment of the invention is a polypeptide having 100% sequenceidentity to the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, or the maturepolypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

The present invention relates to isolated polypeptides having a sequenceidentity to the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, or the maturepolypeptide of SEQ ID NO: 2 or SEQ ID NO: 4 of at least 84%, e.g., atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%, which have protease activity. In one aspect, the polypeptidesdiffer by no more than thirty amino acids, e.g., by twenty five aminoacids, by twenty amino acids, by fifteen amino acids, by twelve aminoacids, by ten amino acids, by nine amino acids, by eight amino acids, byseven amino acids, by six amino acids, by five amino acids, by fouramino acids, by three amino acids, by two amino acids, and by one aminoacid from the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, or the maturepolypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

An embodiment of the invention is a polypeptide having at least 84%sequence identity to the polypeptide of SEQ ID NO: 19, or the maturepolypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide having at least 85%sequence identity to the polypeptide of SEQ ID NO: 19, or the maturepolypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide having at least 86%sequence identity to the polypeptide of SEQ ID NO: 19, or the maturepolypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide having at least 87%sequence identity to the polypeptide of SEQ ID NO: 19, or the maturepolypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide having at least 88%sequence identity to the polypeptide of SEQ ID NO: 19, or the maturepolypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide having at least 89%sequence identity to the polypeptide of SEQ ID NO: 19, or the maturepolypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide having at least 90%sequence identity to the polypeptide of SEQ ID NO: 19, or the maturepolypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide having at least 91%sequence identity to the polypeptide of SEQ ID NO: 19, or the maturepolypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide having at least 92%sequence identity to the polypeptide of SEQ ID NO: 19, or the maturepolypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide having at least 93%sequence identity to the polypeptide of SEQ ID NO: 19, or the maturepolypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide having at least 94%sequence identity to the polypeptide of SEQ ID NO: 19, or the maturepolypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide having at least 95%sequence identity to the polypeptide of SEQ ID NO: 19, or the maturepolypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide having at least 96%sequence identity to the polypeptide of SEQ ID NO: 19, or the maturepolypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide having at least 97%sequence identity to the polypeptide of SEQ ID NO: 19, or the maturepolypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide having at least 98%sequence identity to the polypeptide of SEQ ID NO: 19, or the maturepolypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide having at least 99%sequence identity to the polypeptide of SEQ ID NO: 19, or the maturepolypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide having 100% sequenceidentity to the polypeptide of SEQ ID NO: 19, or the mature polypeptideof SEQ ID NO: 16 or SEQ ID NO: 18.

The present invention relates to isolated polypeptides having a sequenceidentity to the polypeptide of SEQ ID NO: 19, or the mature polypeptideof SEQ ID NO: 16 or SEQ ID NO: 18 of at least 83%, e.g., at least 84%,at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%, which have protease activity. In one aspect, the polypeptidesdiffer by no more than thirty amino acids, e.g., by twenty five aminoacids, by twenty amino acids, by fifteen amino acids, by twelve aminoacids, by ten amino acids, by nine amino acids, by eight amino acids, byseven amino acids, by six amino acids, by five amino acids, by fouramino acids, by three amino acids, by two amino acids, and by one aminoacid from the polypeptide of SEQ ID NO: 19, or the mature polypeptide ofSEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide having at least 86%sequence identity to the polypeptide of SEQ ID NO: 24, or the maturepolypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide having at least 87%sequence identity to the polypeptide of SEQ ID NO: 24, or the maturepolypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide having at least 88%sequence identity to the polypeptide of SEQ ID NO: 24, or the maturepolypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide having at least 89%sequence identity to the polypeptide of SEQ ID NO: 24, or the maturepolypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide having at least 90%sequence identity to the polypeptide of SEQ ID NO: 24, or the maturepolypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide having at least 91%sequence identity to the polypeptide of SEQ ID NO: 24, or the maturepolypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide having at least 92%sequence identity to the polypeptide of SEQ ID NO: 24, or the maturepolypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide having at least 93%sequence identity to the polypeptide of SEQ ID NO: 24, or the maturepolypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide having at least 94%sequence identity to the polypeptide of SEQ ID NO: 24, or the maturepolypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide having at least 95%sequence identity to the polypeptide of SEQ ID NO: 24, or the maturepolypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide having at least 96%sequence identity to the polypeptide of SEQ ID NO: 24, or the maturepolypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide having at least 97%sequence identity to the polypeptide of SEQ ID NO: 24, or the maturepolypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide having at least 98%sequence identity to the polypeptide of SEQ ID NO: 24, or the maturepolypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide having at least 99%sequence identity to the polypeptide of SEQ ID NO: 24, or the maturepolypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide having 100% sequenceidentity to the polypeptide of SEQ ID NO: 24, or the mature polypeptideof SEQ ID NO: 21 or SEQ ID NO: 23.

The present invention relates to isolated polypeptides having a sequenceidentity to the polypeptide of SEQ ID NO: 24, or the mature polypeptideof SEQ ID NO: 21 or SEQ ID NO: 23 of at least 85%, e.g., at least 86%,at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%, which have proteaseactivity. In one aspect, the polypeptides differ by no more than thirtyamino acids, e.g., by twenty five amino acids, by twenty amino acids, byfifteen amino acids, by twelve amino acids, by ten amino acids, by nineamino acids, by eight amino acids, by seven amino acids, by six aminoacids, by five amino acids, by four amino acids, by three amino acids,by two amino acids, and by one amino acid from the polypeptide of SEQ IDNO: 24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

The present invention also relates to the use of isolated polypeptidesin animal feed having protease activity selected from the groupconsisting of:

(a) a polypeptide having at least 60% sequence identity to thepolypeptide of SEQ ID NO: 5, SEQ ID NO: 6, or the mature polypeptide ofSEQ ID NO: 2 or SEQ ID NO: 4;

(b) a polypeptide having at least 60% sequence identity to thepolypeptide of SEQ ID NO: 19, or the mature polypeptide of SEQ ID NO: 16or SEQ ID NO: 18;

(c) a polypeptide having at least 60% sequence identity to thepolypeptide of SEQ ID NO: 24, or the mature polypeptide of SEQ ID NO: 21or SEQ ID NO: 23;

(d) a polypeptide encoded by a polynucleotide that hybridizes undermedium stringency conditions, medium-high stringency conditions, highstringency conditions, or very high stringency conditions with

-   -   (i) the mature polypeptide coding sequence of SEQ ID NO: 1,    -   (ii) the mature polypeptide coding sequence of SEQ ID NO: 3,    -   (iii) the mature polypeptide coding sequence of SEQ ID NO: 15,    -   (iv) the mature polypeptide coding sequence of SEQ ID NO: 17,    -   (v) the mature polypeptide coding sequence of SEQ ID NO: 20,    -   (vi) the mature polypeptide coding sequence of SEQ ID NO: 22,    -   (vii) the full-length complementary strand of (i), (ii), (iii),        (iv), (v) or (vi);

(e) a polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or SEQ ID NO: 3;

(f) a polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 15 or SEQ ID NO: 17;

(g) a polypeptide encoded by a polynucleotide having at least 60%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 20 or SEQ ID NO: 22;

(h) a variant of the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 19 or SEQ ID NO: 24, or the mature polypeptide of SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 21 or SEQ ID NO: 23comprising a substitution, deletion, and/or insertion at one or more(several) positions; and

(i) a fragment of a polypeptide of (a), (b), (c), (d), (e), (f), (g) or(h) having protease activity.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 70% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 75% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 80% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 85% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 87% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 90% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 91% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 92% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 93% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 94% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 95% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 96% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 97% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 98% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 99% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a polypeptide for use in animal feedhaving 100% sequence identity to the polypeptide of SEQ ID NO: 5, SEQ IDNO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

The present invention relates to the use in animal feed of isolatedpolypeptides having a sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4 of at least 60%, e.g., at least 70%, at least 80%, at least 85%, atleast 87%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%, which have protease activity. In one aspect, thepolypeptides differ by no more than twenty amino acids, e.g., by fifteenamino acids, by ten amino acids, by eight amino acids, by seven aminoacids, by six amino acids, by five amino acids, by four amino acids, bythree amino acids, by two amino acids, and by one amino acid from thepolypeptide of SEQ ID NO: 5, SEQ ID NO: 6, or the mature polypeptide ofSEQ ID NO: 2 or SEQ ID NO: 4.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 70% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 75% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 80% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 85% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 87% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 90% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 91% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 92% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 93% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 94% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 95% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 96% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 97% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 98% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 99% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a polypeptide for use in animal feedhaving 100% sequence identity to the polypeptide of SEQ ID NO: 19, orthe mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

The present invention relates to the use in animal feed of isolatedpolypeptides having a sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18 of atleast 60%, e.g., at least 70%, at least 80%, at least 85%, at least 87%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%, which have protease activity. In one aspect, the polypeptidesdiffer by no more than twenty amino acids, e.g., by fifteen amino acids,by ten amino acids, by eight amino acids, by seven amino acids, by sixamino acids, by five amino acids, by four amino acids, by three aminoacids, by two amino acids, and by one amino acid from the polypeptide ofSEQ ID NO: 19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO:18.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 70% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 75% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 80% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 85% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 87% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 90% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 91% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 92% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 93% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 94% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 95% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 96% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 97% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 98% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide for use in animal feedhaving at least 99% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a polypeptide for use in animal feedhaving 100% sequence identity to the polypeptide of SEQ ID NO: 24, orthe mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

The present invention relates to the use in animal feed of isolatedpolypeptides having a sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23 of atleast 60%, e.g., at least 70%, at least 80%, at least 85%, at least 87%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%, which have protease activity. In one aspect, the polypeptidesdiffer by no more than twenty amino acids, e.g., by fifteen amino acids,by ten amino acids, by eight amino acids, by seven amino acids, by sixamino acids, by five amino acids, by four amino acids, by three aminoacids, by two amino acids, and by one amino acid from the polypeptide ofSEQ ID NO: 24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO:23.

In a particular embodiment, the present invention also relates to amethod for preparing an animal feed or feed additive, comprisingpreparing an animal feed or feed additive composition comprising ananimal feed and a protease of selected from the group consisting of:

(a) a polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, or the maturepolypeptide of SEQ ID NO: 2 or SEQ ID NO: 4;

(b) a polypeptide of SEQ ID NO: 19, or the mature polypeptide of SEQ IDNO: 16 or SEQ ID NO: 18;

(c) a polypeptide of SEQ ID NO: 24, or the mature polypeptide of SEQ IDNO: 21 or SEQ ID NO: 23;

(d) a polypeptide having at least 60%, e.g., at least 70%, at least 80%,at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the polypeptide ofSEQ ID NO: 5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 orSEQ ID NO: 4;

(e) a polypeptide having at least 60%, e.g., at least 70%, at least 80%,at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the polypeptide ofSEQ ID NO: 19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO:18; and

(f) a polypeptide having at least 60%, e.g., at least 70%, at least 80%,at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the polypeptide ofSEQ ID NO: 24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO:23.

The present invention also relates to an animal feed or feed additivecomposition comprising an animal feed and a protease of selected fromthe group consisting of:

(a) a polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, or the maturepolypeptide of SEQ ID NO: 2 or SEQ ID NO: 4;

(b) a polypeptide of SEQ ID NO: 19, or the mature polypeptide of SEQ IDNO: 16 or SEQ ID NO: 18;

(c) a polypeptide of SEQ ID NO: 24, or the mature polypeptide of SEQ IDNO: 21 or SEQ ID NO: 23;

(d) a polypeptide having at least 60%, e.g., at least 70%, at least 80%,at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the polypeptide ofSEQ ID NO: 5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 orSEQ ID NO: 4;

(e) a polypeptide having at least 60%, e.g., at least 70%, at least 80%,at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the polypeptide ofSEQ ID NO: 19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO:18; and

(f) a polypeptide having at least 60%, e.g., at least 70%, at least 80%,at least 85%, at least 87%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100% sequence identity to the polypeptide ofSEQ ID NO: 24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO:23.

In one aspect, the polypeptides differ by no more than twenty aminoacids, e.g., by fifteen amino acids, by ten amino acids, by eight aminoacids, by seven amino acids, by six amino acids, by five amino acids, byfour amino acids, by three amino acids, by two amino acids, and by oneamino acid from the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, or themature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

The animal feed compositions may in particular embodiments be in theform of a pellet, a mash or liquid composition, as further describedherein.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 5, SEQ ID NO: 6, or the maturepolypeptide of SEQ ID NO: 2 or SEQ ID NO: 4, an allelic variant thereof;or is a fragment missing, e.g., 30, 25, 20, 15, 10 or 5 amino acids fromthe N- and/or C-terminal and having protease activity. In anotheraspect, the polypeptide comprises or consists of the polypeptide of SEQID NO: 5. In another preferred aspect, the polypeptide comprises orconsists of amino acids 1 to 366 of SEQ ID NO: 2.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 19, or the mature polypeptideof SEQ ID NO: 16 or SEQ ID NO: 18, an allelic variant thereof; or is afragment missing, e.g., 30, 25, 20, 15, 10 or 5 amino acids from the N-and/or C-terminal and having protease activity. In another aspect, thepolypeptide comprises or consists of the polypeptide of SEQ ID NO: 19.In another preferred aspect, the polypeptide comprises or consists ofamino acids 1 to 366 of SEQ ID NO: 16.

A polypeptide of the present invention preferably comprises or consistsof the amino acid sequence of SEQ ID NO: 24, or the mature polypeptideof SEQ ID NO: 21 or SEQ ID NO: 23, an allelic variant thereof; or is afragment missing, e.g., 30, 25, 20, 15, 10 or 5 amino acids from the N-and/or C-terminal and having protease activity. In another aspect, thepolypeptide comprises or consists of the polypeptide of SEQ ID NO: 24.In another preferred aspect, the polypeptide comprises or consists ofamino acids 1 to 366 of SEQ ID NO: 21.

The present invention also relates to isolated polypeptides havingprotease activity that are encoded by polynucleotides that hybridizeunder low stringency conditions, medium stringency conditions,medium-high stringency conditions, high stringency conditions, or veryhigh stringency conditions with (i) the mature polypeptide codingsequence of SEQ ID NO: 1, or (ii) the full-length complementary strandof (i) (J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, MolecularCloning, A Laboratory Manual, 2d edition, Cold Spring Harbor, N.Y.).

The polynucleotide of SEQ ID NO: 1, SEQ ID NO: 15 or SEQ ID NO: 20 or asubsequence thereof, as well as the amino acid sequence of SEQ ID NO: 5,SEQ ID NO: 6, SEQ ID NO: 19, SEQ ID NO: 24, or the mature polypeptide ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 21or SEQ ID NO: 23, or a fragment thereof, may be used to design nucleicacid probes to identify and clone DNA encoding polypeptides havingprotease activity from strains of different genera or species accordingto methods well known in the art. In particular, such probes can be usedfor hybridization with the genomic or cDNA of the genus or species ofinterest, following standard Southern blotting procedures, in order toidentify and isolate the corresponding gene therein. Such probes can beconsiderably shorter than the entire sequence, but should be at least14, e.g., at least 25, at least 35, or at least 70 nucleotides inlength. Preferably, the nucleic acid probe is at least 100 nucleotidesin length, e.g., at least 200 nucleotides, at least 300 nucleotides, atleast 400 nucleotides, at least 500 nucleotides, at least 600nucleotides, at least 700 nucleotides, at least 800 nucleotides, or atleast 900 nucleotides in length. Both DNA and RNA probes can be used.The probes are typically labeled for detecting the corresponding gene(for example, with ³²P, ³H, ³⁵S, biotin, or avidin). Such probes areencompassed by the present invention.

A genomic DNA or cDNA library prepared from such other strains may bescreened for DNA that hybridizes with the probes described above andencodes a polypeptide having protease activity. Genomic or other DNAfrom such other strains may be separated by agarose or polyacrylamidegel electrophoresis, or other separation techniques. DNA from thelibraries or the separated DNA may be transferred to and immobilized onnitrocellulose or other suitable carrier material. In order to identifya clone or DNA that is homologous with SEQ ID NO: 1, SEQ ID NO: 15 orSEQ ID NO: 20 or a subsequence thereof, the carrier material ispreferably used in a Southern blot.

For purposes of the present invention, hybridization indicates that thepolynucleotide hybridizes to a labelled nucleic acid probe correspondingto the mature polypeptide coding sequence of SEQ ID NO: 1, SEQ ID NO: 15or SEQ ID NO: 20, its full-length complementary strand or a subsequencethereof under very low to very high stringency conditions. Molecules towhich the nucleic acid probe hybridizes under these conditions can bedetected using, for example, X-ray film or any other detection meansknown in the art.

In one aspect, the nucleic acid probe is the mature polypeptide codingsequence of SEQ ID NO: 1, SEQ ID NO: 15 or SEQ ID NO: 20. In anotheraspect, the nucleic acid probe is a fragment thereof. In another aspect,the nucleic acid probe is a polynucleotide that encodes the polypeptideof SEQ ID NO: 2, SEQ ID NO: 16, SEQ ID NO: 21 or a fragment thereof. Inanother preferred aspect, the nucleic acid probe is SEQ ID NO: 1, SEQ IDNO: 15 or SEQ ID NO: 20.

For long probes of at least 100 nucleotides in length, high to very highstringency conditions are defined as prehybridization and hybridizationat 42° C. in 5×SSPE, 0.3% SDS, 200 micrograms/ml sheared and denaturedsalmon sperm DNA, and either 25% formamide for very low and lowstringencies, 35% formamide for medium and medium-high stringencies, or50% formamide for high and very high stringencies, following standardSouthern blotting procedures for 12 to 24 hours optimally. The carriermaterial is finally washed three times each for 15 minutes using 2×SSC,0.2% SDS at 65° C. (high stringency), and at 70° C. (very highstringency).

For short probes of about 15 nucleotides to about 70 nucleotides inlength, stringency conditions are defined as prehybridization andhybridization at about 5° C. to about 10° C. below the calculated T_(m)using the calculation according to Bolton and McCarthy (1962, Proc.Natl. Acad. Sci. USA 48:1390) in 0.9 M NaCl, 0.09 M Tris-HCl pH 7.6, 6mM EDTA, 0.5% NP-40, 1×Denhardt's solution, 1 mM sodium pyrophosphate, 1mM sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA perml following standard Southern blotting procedures for 12 to 24 hoursoptimally. The carrier material is finally washed once in 6×SCC plus0.1% SDS for 15 minutes and twice each for 15 minutes using 6×SSC at 5°C. to 10° C. below the calculated T_(m).

The present invention also relates to isolated polypeptides havingprotease activity encoded by polynucleotides having a sequence identityto the mature polypeptide coding sequence of SEQ ID NO: 1 of at least84%, e.g., at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%.

The present invention also relates to isolated polypeptides havingprotease activity encoded by polynucleotides having a sequence identityto the mature polypeptide coding sequence of SEQ ID NO: 15 of at least83%, e.g., at least 84%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%.

The present invention also relates to isolated polypeptides havingprotease activity encoded by polynucleotides having a sequence identityto the mature polypeptide coding sequence of SEQ ID NO: 20 of at least85%, e.g., at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99%, or100%.

In another embodiment, the present invention relates to variantscomprising a substitution, deletion, and/or insertion at one or more(several) positions of SEQ ID NO: 5, SEQ ID NO: 6, or the maturepolypeptide of SEQ ID NO: 2 or SEQ ID NO: 4, or a homologous sequencethereof. The amino acid changes may be of a minor nature, that isconservative amino acid substitutions, insertions or deletions that donot significantly affect the folding and/or activity of the protein;small deletions, typically of one to about 30 amino acids; small amino-or carboxyl-terminal extensions, such as an amino-terminal methionineresidue; a small linker peptide of up to about 20-25 residues; or asmall extension that facilitates purification by changing net charge oranother function, such as a poly-histidine tag or HQ-tag, an antigenicepitope or a binding domain.

In another embodiment, the present invention relates to variantscomprising a substitution, deletion, and/or insertion at one or more(several) positions of SEQ ID NO: 19, or the mature polypeptide of SEQID NO: 16 or SEQ ID NO: 18, or a homologous sequence thereof. The aminoacid changes may be of a minor nature, that is conservative amino acidsubstitutions, insertions or deletions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof one to about 30 amino acids; small amino- or carboxyl-terminalextensions, such as an amino-terminal methionine residue; a small linkerpeptide of up to about 20-25 residues; or a small extension thatfacilitates purification by changing net charge or another function,such as a poly-histidine tag or HQ-tag, an antigenic epitope or abinding domain.

In another embodiment, the present invention relates to variantscomprising a substitution, deletion, and/or insertion at one or more(several) positions of SEQ ID NO: 24, or the mature polypeptide of SEQID NO: 21 or SEQ ID NO: 23, or a homologous sequence thereof. The aminoacid changes may be of a minor nature, that is conservative amino acidsubstitutions, insertions or deletions that do not significantly affectthe folding and/or activity of the protein; small deletions, typicallyof one to about 30 amino acids; small amino- or carboxyl-terminalextensions, such as an amino-terminal methionine residue; a small linkerpeptide of up to about 20-25 residues; or a small extension thatfacilitates purification by changing net charge or another function,such as a poly-histidine tag or HQ-tag, an antigenic epitope or abinding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions that do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges that are expected not to alter the specificactivity substantially are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly,Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn,Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

Alternatively, the amino acid changes are of such a nature that thephysico-chemical properties of the polypeptides are altered. Forexample, amino acid changes may improve the thermal stability of thepolypeptide, alter the substrate specificity, change the pH optimum, andthe like.

Essential amino acids in a parent polypeptide can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for protease activity to identifyamino acid residues that are critical to the activity of the molecule.See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The activesite of the enzyme or other biological interaction can also bedetermined by physical analysis of structure, as determined by suchtechniques as nuclear magnetic resonance, crystallography, electrondiffraction, or photoaffinity labeling, in conjunction with mutation ofputative contact site amino acids. See, for example, de Vos et al.,1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identities ofessential amino acids can also be inferred from analysis of identitieswith polypeptides that are related to the parent polypeptide.

Single or multiple amino acid substitutions, deletions, and/orinsertions can be made and tested using known methods of mutagenesis,recombination, and/or shuffling, followed by a relevant screeningprocedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988,Science 241: 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA86: 2152-2156; WO 95/17413; or WO 95/22625. Other methods that can beused include error-prone PCR, phage display (e.g., Lowman et al., 1991,Biochemistry 30: 10832-10837; U.S. Pat. No. 5,223,409; WO 92/06204), andregion-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Neret al., 1988, DNA 7: 127).

Mutagenesis/shuffling methods can be combined with high-throughput,automated screening methods to detect activity of cloned, mutagenizedpolypeptides expressed by host cells (Ness et al., 1999, NatureBiotechnology 17: 893-896). Mutagenized DNA molecules that encode activepolypeptides can be recovered from the host cells and rapidly sequencedusing standard methods in the art. These methods allow the rapiddetermination of the importance of individual amino acid residues in apolypeptide.

The present invention also relates to variant polypeptides havingprotease activity and having at least 84%, e.g., at least 85%, at least86%, at least 87%, at least 88%, at least 89%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, or at least 99% sequence identity toSEQ ID NO: 5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 orSEQ ID NO: 4 comprising at least one substitution, deletion, and/orinsertion of at least one or more (several) amino acids of SEQ ID NO: 5,SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4or a homologous sequence thereof.

The variant polypeptide of the invention may in one embodiment have atleast 85% sequence identity to SEQ ID NO: 5.

The variant polypeptide of the invention may in one embodiment have atleast 86% sequence identity to SEQ ID NO: 5.

The variant polypeptide of the invention may in one embodiment have atleast 87% sequence identity to SEQ ID NO: 5.

The variant polypeptide of the invention may in one embodiment have atleast 88% sequence identity to SEQ ID NO: 5.

The variant polypeptide of the invention may in one embodiment have atleast 89% sequence identity to SEQ ID NO: 5.

The variant polypeptide of the invention may in one embodiment have atleast 90% sequence identity to SEQ ID NO: 5.

The variant polypeptide of the invention may in one embodiment have atleast 91% sequence identity to SEQ ID NO: 5.

The variant polypeptide of the invention may in one embodiment have atleast 92% sequence identity to SEQ ID NO: 5.

The variant polypeptide of the invention may in one embodiment have atleast 93% sequence identity to SEQ ID NO: 5.

The variant polypeptide of the invention may in one embodiment have atleast 94% sequence identity to SEQ ID NO: 5.

The variant polypeptide of the invention may in one embodiment have atleast 95% sequence identity to SEQ ID NO: 5.

The variant polypeptide of the invention may in one embodiment have atleast 96% sequence identity to SEQ ID NO: 5.

The variant polypeptide of the invention may in one embodiment have atleast 97% sequence identity to SEQ ID NO: 5.

The variant polypeptide of the invention may in one embodiment have atleast 98% sequence identity to SEQ ID NO: 5.

The variant polypeptide of the invention may in one embodiment have atleast 99% sequence identity to SEQ ID NO: 5.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, orthe mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4 is not more than20, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19 or 20.

The present invention also relates to variant polypeptides havingprotease activity and having at least 83%, e.g., at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to SEQ ID NO: 19, or the mature polypeptide of SEQ ID NO: 16 orSEQ ID NO: 18 comprising at least one substitution, deletion, and/orinsertion of at least one or more (several) amino acids of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18 or ahomologous sequence thereof.

The variant polypeptide of the invention may in one embodiment have atleast 84% sequence identity to SEQ ID NO: 19.

The variant polypeptide of the invention may in one embodiment have atleast 85% sequence identity to SEQ ID NO: 19.

The variant polypeptide of the invention may in one embodiment have atleast 86% sequence identity to SEQ ID NO: 19.

The variant polypeptide of the invention may in one embodiment have atleast 87% sequence identity to SEQ ID NO: 19.

The variant polypeptide of the invention may in one embodiment have atleast 88% sequence identity to SEQ ID NO: 19.

The variant polypeptide of the invention may in one embodiment have atleast 89% sequence identity to SEQ ID NO: 19.

The variant polypeptide of the invention may in one embodiment have atleast 90% sequence identity to SEQ ID NO: 19.

The variant polypeptide of the invention may in one embodiment have atleast 91% sequence identity to SEQ ID NO: 19.

The variant polypeptide of the invention may in one embodiment have atleast 92% sequence identity to SEQ ID NO: 19.

The variant polypeptide of the invention may in one embodiment have atleast 93% sequence identity to SEQ ID NO: 19.

The variant polypeptide of the invention may in one embodiment have atleast 94% sequence identity to SEQ ID NO: 19.

The variant polypeptide of the invention may in one embodiment have atleast 95% sequence identity to SEQ ID NO: 19.

The variant polypeptide of the invention may in one embodiment have atleast 96% sequence identity to SEQ ID NO: 19.

The variant polypeptide of the invention may in one embodiment have atleast 97% sequence identity to SEQ ID NO: 19.

The variant polypeptide of the invention may in one embodiment have atleast 98% sequence identity to SEQ ID NO: 19.

The variant polypeptide of the invention may in one embodiment have atleast 99% sequence identity to SEQ ID NO: 19.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 19, or the maturepolypeptide of SEQ ID NO: 16 or SEQ ID NO: 18 is not more than 20, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

The present invention also relates to variant polypeptides havingprotease activity and having at least 85%, e.g., at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 24,or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23 comprisingat least one substitution, deletion, and/or insertion of at least one ormore (several) amino acids of SEQ ID NO: 24, or the mature polypeptideof SEQ ID NO: 21 or SEQ ID NO: 23 or a homologous sequence thereof.

The variant polypeptide of the invention may in one embodiment have atleast 86% sequence identity to SEQ ID NO: 24.

The variant polypeptide of the invention may in one embodiment have atleast 87% sequence identity to SEQ ID NO: 24.

The variant polypeptide of the invention may in one embodiment have atleast 88% sequence identity to SEQ ID NO: 24.

The variant polypeptide of the invention may in one embodiment have atleast 89% sequence identity to SEQ ID NO: 24.

The variant polypeptide of the invention may in one embodiment have atleast 90% sequence identity to SEQ ID NO: 24.

The variant polypeptide of the invention may in one embodiment have atleast 91% sequence identity to SEQ ID NO: 24.

The variant polypeptide of the invention may in one embodiment have atleast 92% sequence identity to SEQ ID NO: 24.

The variant polypeptide of the invention may in one embodiment have atleast 93% sequence identity to SEQ ID NO: 24.

The variant polypeptide of the invention may in one embodiment have atleast 94% sequence identity to SEQ ID NO: 24.

The variant polypeptide of the invention may in one embodiment have atleast 95% sequence identity to SEQ ID NO: 24.

The variant polypeptide of the invention may in one embodiment have atleast 96% sequence identity to SEQ ID NO: 24.

The variant polypeptide of the invention may in one embodiment have atleast 97% sequence identity to SEQ ID NO: 24.

The variant polypeptide of the invention may in one embodiment have atleast 98% sequence identity to SEQ ID NO: 24.

The variant polypeptide of the invention may in one embodiment have atleast 99% sequence identity to SEQ ID NO: 24.

The total number of amino acid substitutions, deletions and/orinsertions of the mature polypeptide of SEQ ID NO: 24, or the maturepolypeptide of SEQ ID NO: 21 or SEQ ID NO: 23 is not more than 20, e.g.,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.

The polypeptide may be hybrid polypeptide in which a portion of onepolypeptide is fused at the N-terminus or the C-terminus of a portion ofanother polypeptide.

The polypeptide may be a fusion polypeptide or cleavable fusionpolypeptide in which another polypeptide is fused at the N-terminus orthe C-terminus of the polypeptide of the present invention. A fusedpolypeptide is produced by fusing a polynucleotide encoding anotherpolypeptide to a polynucleotide of the present invention. Techniques forproducing fusion polypeptides are known in the art, and include ligatingthe coding sequences encoding the polypeptides so that they are in frameand that expression of the fused polypeptide is under control of thesame promoter(s) and terminator. Fusion proteins may also be constructedusing intein technology in which fusions are createdpost-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawsonet al., 1994, Science 266: 776-779).

A fusion polypeptide can further comprise a cleavage site between thetwo polypeptides. Upon secretion of the fusion protein, the site iscleaved releasing the two polypeptides. Examples of cleavage sitesinclude, but are not limited to, the sites disclosed in Martin et al.,2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000,J. Biotechnol. 76: 245-251; Rasmussen-Wilson et al., 1997, Appl.Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13:498-503; and Contreras et al., 1991, Biotechnology 9: 378-381; Eaton etal., 1986, Biochemistry 25: 505-512; Collins-Racie et al., 1995,Biotechnology 13: 982-987; Carter et al., 1989, Proteins: Structure,Function, and Genetics 6: 240-248; and Stevens, 2003, Drug DiscoveryWorld 4: 35-48.

The polypeptide may be expressed by a recombinant DNA sequencecontaining the coding for a His-tag or HQ-tag to give, after anypost-translational modifications, the mature polypeptide containing allor part of the His- or HQ-tag. The HQ-tag, having the sequence—RHQHQHQ,may be fully or partly cleaved off the polypeptide during thepost-translational modifications resulting in for example the additionalamino acids-RHQHQ attached to the C-terminal of the mature polypeptide.

Carbohydrate molecules are often attached to a polypeptide from a fungalsource during post-translational modification. In order to aid massspectrometry analysis, the polypeptide can be incubated with anendoglycosidase to deglycosylate each N-linked position. For everydeglycosylated N-linked site, one N-acetyl hexosamine remains on theprotein backbone.

EMBODIMENTS

In certain embodiments of the invention, the protease of the inventionexhibits beneficial thermal properties such as thermostability, steamstability, etc and/or pH properties, such as acid stability, pH optimum,etc.

An embodiment of the invention is isolated polypeptides having improvedprotease activity between pH 2 and 5, such as between pH 2 and 4,preferably between pH 3 and 5, or more preferably between pH 3 and 4, at25° C. compared to protease 10R.

A further embodiment of the invention is isolated polypeptides havingimproved protease activity at, e.g., 60° C. or below, preferably 50° C.or below, more preferably 37° C. or below; between 25° C. and 60° C.,preferably between 25° C. and 50° C.; or at 25° C. or at 37° C. comparedto protease 10R.

An additional embodiment of the invention is improved protease activityon soybean-maze meal between pH 3.0 and 4.0 at 40° C. compared toprotease 10R.

Another embodiment of the invention is improved proteolytic activity onbroiler digesta expressed as increase in level of primary amines in cropand/or gizzard digesta after 3 or 1 hour incubation when compared to anon-protease treated blank sample and when compared to a sample treatedwith protease 10R.

Acidity/Alkalinity Properties

In certain embodiments of the invention the protease of the inventionexhibits beneficial properties in respect of pH, such as acid stability,pH optimum, etc. Stability of the protease at a low pH is beneficialsince the protease can have activity in the intestine after passingthrough the stomach. In one embodiment of the invention, the proteaseretains >70% activity, such as >95% activity after 2 hours at pH 3 asdetermined using the method described in Example 3.

Temperature-Activity

The temperature-activity profile of the protease may be determined asdescribed in Example 3. Activity at low temperatures (30-40° C.) can beadvantageous for the digestion of proteins in an animal.

In one embodiment, the invention comprises of a protease having atemperature activity profile at pH 4.0 with relative activity of 0.20 orhigher at 25° C., or relative activity of 0.50 or higher at 37° C. whencompared to the activity of the protease at 50° C. (cf. Example 3).

Thermostability

Thermostability may be determined as described in Example 6, i.e., usingDSC measurements to determine the denaturation temperature, T_(d), ofthe purified protease protein. The Td is indicative of thethermostability of the protein: The higher the T_(d), the higher thethermostability. Accordingly, in a preferred embodiment, the protease ofthe invention has a T_(d) which is higher than the T_(d) of a referenceprotease, wherein T_(d) is determined on purified protease samples(preferably with a purity of at least 90% or 95%, as determined bySDS-PAGE).

In preferred embodiments, the thermal properties such as heat-stability,temperature stability, thermostability, steam stability, and/orpelleting stability as provided by the residual activity, denaturationtemperature T_(d), or other parameter of the protease of the inventionis higher than the corresponding value, such as the residual activity orT_(d), of the protease of SEQ ID NO: 6, more preferably at least 101%thereof, or at least 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, orat least 110% thereof. Even more preferably, the value of the parameter,such as residual activity or T_(d), of the protease of the invention isat least 120%, 130%, 140%, 150%, 160%, 170%, 180%, or at least 190% ofthe value for the protease of SEQ ID NO: 6.

In preferred embodiments, the thermal properties such as heat-stability,temperature stability, thermostability, steam stability, and/orpelleting stability as provided by the residual activity, denaturationtemperature T_(d), or other parameter of the protease of the inventionis higher than the corresponding value, such as the residual activity orT_(d), of the protease of SEQ ID NO: 19, more preferably at least 101%thereof, or at least 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, orat least 110% thereof. Even more preferably, the value of the parameter,such as residual activity or T_(d), of the protease of the invention isat least 120%, 130%, 140%, 150%, 160%, 170%, 180%, or at least 190% ofthe value for the protease of SEQ ID NO: 19.

In preferred embodiments, the thermal properties such as heat-stability,temperature stability, thermostability, steam stability, and/orpelleting stability as provided by the residual activity, denaturationtemperature T_(d), or other parameter of the protease of the inventionis higher than the corresponding value, such as the residual activity orT_(d), of the protease of SEQ ID NO: 24, more preferably at least 101%thereof, or at least 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, orat least 110% thereof. Even more preferably, the value of the parameter,such as residual activity or T_(d), of the protease of the invention isat least 120%, 130%, 140%, 150%, 160%, 170%, 180%, or at least 190% ofthe value for the protease of SEQ ID NO: 24.

In still further particular embodiments, the thermostable protease ofthe invention has a melting temperature, T_(m) (or a denaturationtemperature, T_(d)), as determined using Differential Scanningcalorimetry (DSC) as described in example 10 (i.e., in 20 mM sodiumacetate, pH 4.0), of at least 50° C. In still further particularembodiments, the T_(m) is at least 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99 or at least 100° C.

Steam Stability

Steam stability may be determined as described in Example 7 bydetermining the residual activity of protease molecules after steamtreatment at 85° C. or 90° C. for a short time.

Pelleting Stability

Pelleting stability may be determined as described in Example 8 by usingenzyme granulate pre-mixed with feed. From the mixer the feed isconditioned with steam to 95° C. After conditioning the feed is pressedto pellets and the residual activity determined.

Sources of Polypeptides Having Protease Activity

A polypeptide having protease activity of the present invention may beobtained from fungi of any genus. For purposes of the present invention,the term “obtained from” as used herein in connection with a givensource shall mean that the polypeptide encoded by a polynucleotide isproduced by the source or by a strain in which the polynucleotide fromthe source has been inserted. In one aspect, the polypeptide obtainedfrom a given source is secreted extracellularly.

The polypeptide may be a fungal polypeptide. For example, thepolypeptide may be a polypeptide having protease activity from within aphylum such as Basidiomycota. In one aspect, the polypeptide is aprotease from a fungus of the class Agaricomycetes, such as from theorder Polyporales, or from the family Coriolaceae, or from the genusMeripilus or from the genus Trametes.

It will be understood that for the aforementioned species, the inventionencompasses both the perfect and imperfect states, and other taxonomicequivalents, e.g., anamorphs, regardless of the species name by whichthey are known. Those skilled in the art will readily recognize theidentity of appropriate equivalents.

Strains of these taxa are readily accessible to the public in a numberof culture collections, such as the American Type Culture Collection(ATCC), Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH(DSMZ), Centraalbureau Voor Schimmelcultures (CBS), and AgriculturalResearch Service Patent Culture Collection, Northern Regional ResearchCenter (NRRL).

The polypeptide may be identified and obtained from other sourcesincluding microorganisms isolated from nature (e.g., soil, composts,water, etc.) or DNA samples obtained directly from natural materials(e.g., soil, composts, water, etc.) using the above-mentioned probes.Techniques for isolating microorganisms and DNA directly from naturalhabitats are well known in the art. A polynucleotide encoding thepolypeptide may then be obtained by similarly screening a genomic DNA orcDNA library of another microorganism or mixed DNA sample. Once apolynucleotide encoding a polypeptide has been detected with theprobe(s), the polynucleotide can be isolated or cloned by utilizingtechniques that are known to those of ordinary skill in the art (see,e.g., Sambrook et al., 1989, supra).

Polynucleotides

The present invention also relates to isolated polynucleotides encodinga polypeptide of the present invention.

The techniques used to isolate or clone a polynucleotide encoding apolypeptide are known in the art and include isolation from genomic DNA,preparation from cDNA, or a combination thereof. The cloning of thepolynucleotides from such genomic DNA can be effected, e.g., by usingthe well-known polymerase chain reaction (PCR) or antibody screening ofexpression libraries to detect cloned DNA fragments with sharedstructural features. See, e.g., Innis et al., 1990, PCR: A Guide toMethods and Application, Academic Press, New York. Other nucleic acidamplification procedures such as ligase chain reaction (LCR), ligationactivated transcription (LAT) and polynucleotide-based amplification(NASBA) may be used. The polynucleotides may be cloned from a strain ofBacillus sp., or another or related organism from the order Bacillalesand thus, for example, may be an allelic or species variant of thepolypeptide encoding region of the polynucleotide.

The present invention also relates to isolated polynucleotidescomprising or consisting of polynucleotides having a degree of sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 1 of atleast 84%, e.g., at least 85%, at least 86%, at least 87%, at least 88%,at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%, which encode a polypeptide having protease activity.

The present invention also relates to isolated polynucleotidescomprising or consisting of polynucleotides having a degree of sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 15 ofat least 83%, e.g., at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, at least 99%, or 100%, which encode a polypeptidehaving protease activity.

The present invention also relates to isolated polynucleotidescomprising or consisting of polynucleotides having a degree of sequenceidentity to the mature polypeptide coding sequence of SEQ ID NO: 20 ofat least 85%, e.g., at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least99%, or 100%, which encode a polypeptide having protease activity.

Modification of a polynucleotide encoding a polypeptide of the presentinvention may be necessary for the synthesis of polypeptidessubstantially similar to the polypeptide. The term “substantiallysimilar” to the polypeptide refers to non-naturally occurring forms ofthe polypeptide. These polypeptides may differ in some engineered wayfrom the polypeptide isolated from its native source, e.g., variantsthat differ in specific activity, thermostability, pH optimum, or thelike. The variant may be constructed on the basis of the polynucleotidepresented as the mature polypeptide coding sequence of SEQ ID NO: 1, SEQID NO: 15 or SEQ ID NO: 20, e.g., a subsequence thereof, and/or byintroduction of nucleotide substitutions that do not result in a changein the amino acid sequence of the polypeptide, but which correspond tothe codon usage of the host organism intended for production of theenzyme, or by introduction of nucleotide substitutions that may giverise to a different amino acid sequence. For a general description ofnucleotide substitution, see, e.g., Ford et al., 1991, ProteinExpression and Purification 2: 95-107.

The present invention also relates to isolated polynucleotides encodingpolypeptides of the present invention, which hybridize under very lowstringency conditions, low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 1, (ii) the genomic DNAsequence comprising the mature polypeptide coding sequence of SEQ ID NO:1, or (iii) the full-length complementary strand of (i) or (ii); orallelic variants and subsequences thereof (Sambrook et al., 1989,supra), as defined herein.

In one aspect, the polynucleotide comprises or consists of SEQ ID NO: 1,the mature polypeptide coding sequence of SEQ ID NO: 1, or a subsequenceof SEQ ID NO: 1 that encodes a fragment of SEQ ID NO: 2 having proteaseactivity, such as the polynucleotide of nucleotides 605 to 1702 of SEQID NO: 1.

The present invention also relates to isolated polynucleotides encodingpolypeptides of the present invention, which hybridize under very lowstringency conditions, low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 15, (ii) the genomic DNAsequence comprising the mature polypeptide coding sequence of SEQ ID NO:15, or (iii) the full-length complementary strand of (i) or (ii); orallelic variants and subsequences thereof (Sambrook et al., 1989,supra), as defined herein.

In one aspect, the polynucleotide comprises or consists of SEQ ID NO:15, the mature polypeptide coding sequence of SEQ ID NO: 15, or asubsequence of SEQ ID NO: 15 that encodes a fragment of SEQ ID NO: 16having protease activity, such as the joined sequence of nucleotides1207 to 1353, nucleotides 1412 to 1522, nucleotides 1577 to 1776,nucleotides 1832 to 1969, nucleotides 2031 to 2478 and nucleotides 2531to 2584 of SEQ ID NO: 15.

The present invention also relates to isolated polynucleotides encodingpolypeptides of the present invention, which hybridize under very lowstringency conditions, low stringency conditions, medium stringencyconditions, medium-high stringency conditions, high stringencyconditions, or very high stringency conditions with (i) the maturepolypeptide coding sequence of SEQ ID NO: 20, (ii) the genomic DNAsequence comprising the mature polypeptide coding sequence of SEQ ID NO:20, or (iii) the full-length complementary strand of (i) or (ii); orallelic variants and subsequences thereof (Sambrook et al., 1989,supra), as defined herein.

In one aspect, the polynucleotide comprises or consists of SEQ ID NO:20, the mature polypeptide coding sequence of SEQ ID NO: 20, or asubsequence of SEQ ID NO: 20 that encodes a fragment of SEQ ID NO: 21having protease activity, such as the joined sequence of nucleotides1206 to 1352, nucleotides 1414 to 1524, nucleotides 1580 to 1779,nucleotides 1833 to 1970, nucleotides 2032 to 2479 and nucleotides 2532to 2585 of SEQ ID NO: 20.

Nucleic Acid Constructs

The present invention also relates to nucleic acid constructs comprisinga polynucleotide of the present invention operably linked to one or more(several) control sequences that direct the expression of the codingsequence in a suitable host cell under conditions compatible with thecontrol sequences.

A polynucleotide may be manipulated in a variety of ways to provide forexpression of the polypeptide. Manipulation of the polynucleotide priorto its insertion into a vector may be desirable or necessary dependingon the expression vector. The techniques for modifying polynucleotidesutilizing recombinant DNA methods are well known in the art.

The control sequence may be a promoter sequence, a polynucleotide thatis recognized by a host cell for expression of a polynucleotide encodinga polypeptide of the present invention. The promoter sequence containstranscriptional control sequences that mediate the expression of thepolypeptide. The promoter may be any polynucleotide that showstranscriptional activity in the host cell of choice including mutant,truncated, and hybrid promoters, and may be obtained from genes encodingextracellular or intracellular polypeptides either homologous orheterologous to the host cell.

Examples of suitable promoters for directing transcription of thenucleic acid constructs of the present invention in a bacterial hostcell are the promoters obtained from the Bacillus amyloliquefaciensalpha-amylase gene (amyQ), Bacillus licheniformis alpha-amylase gene(amyL), Bacillus licheniformis penicillinase gene (penP), Bacillusstearothermophilus maltogenic amylase gene (amyM), Bacillus subtilislevansucrase gene (sacB), Bacillus subtilis xylA and xylB genes,Bacillus thuringiensis cryIIIA gene (Agaisse and Lereclus, 1994,Molecular Microbiology 13: 97-107), E. coli lac operon, E. coli trcpromoter (Egon et al., 1988, Gene 69: 301-315), Streptomyces coelicoloragarase gene (dagA), and prokaryotic beta-lactamase gene (Villa-Kamaroffet al., 1978, Proc. Natl. Acad. Sci. USA 75: 3727-3731), as well as thetac promoter (DeBoer et al., 1983, Proc. Natl. Acad. Sci. USA 80:21-25). Further promoters are described in “Useful proteins fromrecombinant bacteria” in Gilbert et al., 1980, Scientific American 242:74-94; and in Sambrook et al., 1989, supra. Examples of tandem promotersare disclosed in WO 99/43835.

Examples of suitable promoters for directing the transcription of thenucleic acid constructs of the present invention in a filamentous fungalhost cell are promoters obtained from the genes for Aspergillus nidulansacetamidase, Aspergillus niger neutral alpha-amylase, Aspergillus nigeracid stable alpha-amylase, Aspergillus niger or Aspergillus awamoriglucoamylase (glaA), Aspergillus oryzae TAKA amylase, Aspergillus oryzaealkaline protease, Aspergillus oryzae triose phosphate isomerase,Fusarium oxysporum trypsin-like protease (WO 96/00787), Fusariumvenenatum amyloglucosidase (WO 00/56900), Fusarium venenatum Daria (WO00/56900), Fusarium venenatum Quinn (WO 00/56900), Rhizomucor mieheilipase, Rhizomucor miehei aspartic proteinase, Trichoderma reeseibeta-glucosidase, Trichoderma reesei cellobiohydrolase I, Trichodermareesei cellobiohydrolase II, Trichoderma reesei endoglucanase I,Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanaseIII, Trichoderma reesei endoglucanase IV, Trichoderma reeseiendoglucanase V, Trichoderma reesei xylanase I, Trichoderma reeseixylanase II, Trichoderma reesei beta-xylosidase, as well as the NA2-tpipromoter (a modified promoter including a gene encoding a neutralalpha-amylase in Aspergilli in which the untranslated leader has beenreplaced by an untranslated leader from a gene encoding triose phosphateisomerase in Aspergilli; non-limiting examples include modifiedpromoters including the gene encoding neutral alpha-amylase inAspergillus niger in which the untranslated leader has been replaced byan untranslated leader from the gene encoding triose phosphate isomerasein Aspergillus nidulans or Aspergillus oryzae); and mutant, truncated,and hybrid promoters thereof.

In a yeast host, useful promoters are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiaegalactokinase (GAL1), Saccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH1, ADH2/GAP),Saccharomyces cerevisiae triose phosphate isomerase (TPI), Saccharomycescerevisiae metallothionein (CUP1), and Saccharomyces cerevisiae3-phosphoglycerate kinase. Other useful promoters for yeast host cellsare described by Romanos et al., 1992, Yeast 8: 423-488.

The control sequence may also be a suitable transcription terminatorsequence, which is recognized by a host cell to terminate transcription.The terminator sequence is operably linked to the 3′-terminus of thepolynucleotide encoding the polypeptide. Any terminator that isfunctional in the host cell of choice may be used in the presentinvention.

Preferred terminators for bacterial host cells are obtained from thegenes for Bacillus clausii alkaline protease (aprH), Bacilluslicheniformis alpha-amylase (amyL), and Escherichia coli ribosomal RNA(rrnB).

Preferred terminators for filamentous fungal host cells are obtainedfrom the genes for Aspergillus nidulans anthranilate synthase,Aspergillus niger glucoamylase, Aspergillus niger alpha-glucosidase,Aspergillus oryzae TAKA amylase, and Fusarium oxysporum trypsin-likeprotease.

Preferred terminators for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae enolase, Saccharomyces cerevisiaecytochrome C (CYC1), and Saccharomyces cerevisiaeglyceraldehyde-3-phosphate dehydrogenase. Other useful terminators foryeast host cells are described by Romanos et al., 1992, supra.

The control sequence may also be an mRNA stabilizer region downstream ofa promoter and upstream of the coding sequence of a gene which increasesexpression of the gene.

Examples of suitable mRNA stabilizer regions are obtained from aBacillus thuringiensis cryIIIA gene (WO 94/25612) and a Bacillussubtilis SP82 gene (Hue et al., 1995, Journal of Bacteriology 177:3465-3471).

The control sequence may also be a leader, a nontranslated region of anmRNA that is important for translation by the host cell. The leader isoperably linked to the 5′-terminus of the polynucleotide encoding thepolypeptide. Any leader that is functional in the host cell may be used.

Preferred leaders for filamentous fungal host cells are obtained fromthe genes for Aspergillus oryzae TAKA amylase and Aspergillus nidulanstriose phosphate isomerase.

Suitable leaders for yeast host cells are obtained from the genes forSaccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae3-phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, andSaccharomyces cerevisiae alcoholdehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP).

The control sequence may also be a polyadenylation sequence, a sequenceoperably linked to the 3′-terminus of the polynucleotide and, whentranscribed, is recognized by the host cell as a signal to addpolyadenosine residues to transcribed mRNA. Any polyadenylation sequencethat is functional in the host cell of choice may be used.

Preferred polyadenylation sequences for filamentous fungal host cellsare obtained from the genes for Aspergillus oryzae TAKA amylase,Aspergillus niger glucoamylase, Aspergillus nidulans anthranilatesynthase, Fusarium oxysporum trypsin-like protease, and Aspergillusniger alpha-glucosidase.

Useful polyadenylation sequences for yeast host cells are described byGuo and Sherman, 1995, Mol. Cellular Biol. 15: 5983-5990.

The control sequence may also be a signal peptide coding region thatencodes a signal peptide linked to the N-terminus of a polypeptide anddirects the polypeptide into the cell's secretory pathway. The 5′-end ofthe coding sequence of the polynucleotide may inherently contain asignal peptide coding sequence naturally linked in translation readingframe with the segment of the coding sequence that encodes thepolypeptide. Alternatively, the 5′-end of the coding sequence maycontain a signal peptide coding sequence that is foreign to the codingsequence. The foreign signal peptide coding sequence may be requiredwhere the coding sequence does not naturally contain a signal peptidecoding sequence. Alternatively, the foreign signal peptide codingsequence may simply replace the natural signal peptide coding sequencein order to enhance secretion of the polypeptide. However, any signalpeptide coding sequence that directs the expressed polypeptide into thesecretory pathway of a host cell of choice may be used.

Effective signal peptide coding sequences for bacterial host cells arethe signal peptide coding sequences obtained from the genes for BacillusNCIB 11837 maltogenic amylase, Bacillus licheniformis subtilisin,Bacillus licheniformis beta-lactamase, Bacillus stearothermophilusalpha-amylase, Bacillus stearothermophilus neutral proteases (nprT,nprS, nprM), Bacillus subtilis prsA and Bascillus lentus. Further signalpeptides are described by Simonen and Palva, 1993, MicrobiologicalReviews 57: 109-137.

Effective signal peptide coding sequences for filamentous fungal hostcells are the signal peptide coding sequences obtained from the genesfor Aspergillus niger neutral amylase, Aspergillus niger glucoamylase,Aspergillus oryzae TAKA amylase, Humicola insolens cellulase, Humicolainsolens endoglucanase V, Humicola lanuginosa lipase, and Rhizomucormiehei aspartic proteinase.

Useful signal peptides for yeast host cells are obtained from the genesfor Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiaeinvertase. Other useful signal peptide coding sequences are described byRomanos et al., 1992, supra.

The control sequence may also be a propeptide coding sequence thatencodes a propeptide positioned at the N-terminus of a polypeptide. Theresultant polypeptide is known as a proenzyme or propolypeptide (or azymogen in some cases). A propolypeptide is generally inactive and canbe converted to an active polypeptide by catalytic or autocatalyticcleavage of the propeptide from the propolypeptide. The propeptidecoding sequence may be obtained from the genes for Bacillus subtilisalkaline protease (aprE), Bacillus subtilis neutral protease (nprT),Myceliophthora thermophila laccase (WO 95/33836), Rhizomucor mieheiaspartic proteinase, and Saccharomyces cerevisiae alpha-factor.

Where both signal peptide and propeptide sequences are present at theN-terminus of a polypeptide, the propeptide sequence is positioned nextto the N-terminus of a polypeptide and the signal peptide sequence ispositioned next to the N-terminus of the propeptide sequence.

It may also be desirable to add regulatory sequences that allow theregulation of the expression of the polypeptide relative to the growthof the host cell. Examples of regulatory systems are those that causethe expression of the gene to be turned on or off in response to achemical or physical stimulus, including the presence of a regulatorycompound. Regulatory systems in prokaryotic systems include the lac,tac, and trp operator systems. In yeast, the ADH2 system or GAL1 systemmay be used. In filamentous fungi, the Aspergillus niger glucoamylasepromoter, Aspergillus oryzae TAKA alpha-amylase promoter, andAspergillus oryzae glucoamylase promoter may be used. Other examples ofregulatory sequences are those that allow for gene amplification. Ineukaryotic systems, these regulatory sequences include the dihydrofolatereductase gene that is amplified in the presence of methotrexate, andthe metallothionein genes that are amplified with heavy metals. In thesecases, the polynucleotide encoding the polypeptide would be operablylinked with the regulatory sequence.

Expression Vectors

The present invention also relates to recombinant expression vectorscomprising a polynucleotide of the present invention, a promoter, andtranscriptional and translational stop signals. The various nucleotideand control sequences may be joined together to produce a recombinantexpression vector that may include one or more (several) convenientrestriction sites to allow for insertion or substitution of thepolynucleotide encoding the polypeptide at such sites. Alternatively,the polynucleotide may be expressed by inserting the polynucleotide or anucleic acid construct comprising the sequence into an appropriatevector for expression. In creating the expression vector, the codingsequence is located in the vector so that the coding sequence isoperably linked with the appropriate control sequences for expression.

The recombinant expression vector may be any vector (e.g., a plasmid orvirus) that can be conveniently subjected to recombinant DNA proceduresand can bring about expression of the polynucleotide. The choice of thevector will typically depend on the compatibility of the vector with thehost cell into which the vector is to be introduced. The vector may be alinear or closed circular plasmid.

The vector may be an autonomously replicating vector, i.e., a vectorthat exists as an extrachromosomal entity, the replication of which isindependent of chromosomal replication, e.g., a plasmid, anextrachromosomal element, a minichromosome, or an artificial chromosome.The vector may contain any means for assuring self-replication.Alternatively, the vector may be one that, when introduced into the hostcell, is integrated into the genome and replicated together with thechromosome(s) into which it has been integrated. Furthermore, a singlevector or plasmid or two or more vectors or plasmids that togethercontain the total DNA to be introduced into the genome of the host cell,or a transposon, may be used.

The vector preferably contains one or more (several) selectable markersthat permit easy selection of transformed, transfected, transduced, orthe like cells. A selectable marker is a gene the product of whichprovides for biocide or viral resistance, resistance to heavy metals,prototrophy to auxotrophs, and the like.

Examples of bacterial selectable markers are Bacillus licheniformis orBacillus subtilis dal genes, or markers that confer antibioticresistance such as ampicillin, chloramphenicol, kanamycin, neomycin,spectinomycin, or tetracycline resistance. Suitable markers for yeasthost cells include, but are not limited to, ADE2, HIS3, LEU2, LYS2,MET3, TRP1, and URA3. Selectable markers for use in a filamentous fungalhost cell include, but are not limited to, amdS (acetamidase), argB(ornithine carbamoyltransferase), bar (phosphinothricinacetyltransferase), hph (hygromycin phosphotransferase), niaD (nitratereductase), pyrG (orotidine-5′-phosphate decarboxylase), sC (sulfateadenyltransferase), and trpC (anthranilate synthase), as well asequivalents thereof. Preferred for use in an Aspergillus cell areAspergillus nidulans or Aspergillus oryzae amdS and pyrG genes and aStreptomyces hygroscopicus bar gene.

The vector preferably contains an element(s) that permits integration ofthe vector into the host cell's genome or autonomous replication of thevector in the cell independent of the genome.

For integration into the host cell genome, the vector may rely on thepolynucleotide's sequence encoding the polypeptide or any other elementof the vector for integration into the genome by homologous ornon-homologous recombination. Alternatively, the vector may containadditional polynucleotides for directing integration by homologousrecombination into the genome of the host cell at a precise location(s)in the chromosome(s). To increase the likelihood of integration at aprecise location, the integrational elements should contain a sufficientnumber of nucleic acids, such as 100 to 10,000 base pairs, 400 to 10,000base pairs, and 800 to 10,000 base pairs, which have a high degree ofsequence identity to the corresponding target sequence to enhance theprobability of homologous recombination. The integrational elements maybe any sequence that is homologous with the target sequence in thegenome of the host cell. Furthermore, the integrational elements may benon-encoding or encoding polynucleotides. On the other hand, the vectormay be integrated into the genome of the host cell by non-homologousrecombination.

For autonomous replication, the vector may further comprise an origin ofreplication enabling the vector to replicate autonomously in the hostcell in question. The origin of replication may be any plasmidreplicator mediating autonomous replication that functions in a cell.The term “origin of replication” or “plasmid replicator” means apolynucleotide that enables a plasmid or vector to replicate in vivo.

Examples of bacterial origins of replication are the origins ofreplication of plasmids pBR322, pUC19, pACYC177, and pACYC184 permittingreplication in E. coli, and pUB110, pE194, pTA1060, and pAMβ1 permittingreplication in Bacillus.

Examples of origins of replication for use in a yeast host cell are the2 micron origin of replication, ARS1, ARS4, the combination of ARS1 andCEN3, and the combination of ARS4 and CEN6.

Examples of origins of replication useful in a filamentous fungal cellare AMA1 and ANSI (Gems et al., 1991, Gene 98: 61-67; Cullen et al.,1987, Nucleic Acids Res. 15: 9163-9175; WO 00/24883). Isolation of theAMA1 gene and construction of plasmids or vectors comprising the genecan be accomplished according to the methods disclosed in WO 00/24883.

More than one copy of a polynucleotide of the present invention may beinserted into a host cell to increase production of a polypeptide. Anincrease in the copy number of the polynucleotide can be obtained byintegrating at least one additional copy of the sequence into the hostcell genome or by including an amplifiable selectable marker gene withthe polynucleotide where cells containing amplified copies of theselectable marker gene, and thereby additional copies of thepolynucleotide, can be selected for by cultivating the cells in thepresence of the appropriate selectable agent.

The procedures used to ligate the elements described above to constructthe recombinant expression vectors of the present invention are wellknown to one skilled in the art (see, e.g., Sambrook et al., 1989,supra).

Host Cells

The present invention also relates to recombinant host cells, comprisinga polynucleotide of the present invention operably linked to one or more(several) control sequences that direct the production of a polypeptideof the present invention. A construct or vector comprising apolynucleotide is introduced into a host cell so that the construct orvector is maintained as a chromosomal integrant or as a self-replicatingextra-chromosomal vector as described earlier. The term “host cell”encompasses any progeny of a parent cell that is not identical to theparent cell due to mutations that occur during replication. The choiceof a host cell will to a large extent depend upon the gene encoding thepolypeptide and its source.

The host cell may be any cell useful in the recombinant production of apolypeptide of the present invention, e.g., a prokaryote or a eukaryote.

The prokaryotic host cell may be any Gram-positive or Gram-negativebacterium. Gram-positive bacteria include, but are not limited to,Bacillus, Clostridium, Enterococcus, Geobacillus, Lactobacillus,Lactococcus, Oceanobacillus, Staphylococcus, Streptococcus, andStreptomyces. Gram-negative bacteria include, but are not limited to,Campylobacter, E. coli, Flavobacterium, Fusobacterium, Helicobacter,Ilyobacter, Neisseria, Pseudomonas, Salmonella, and Ureaplasma.

The bacterial host cell may be any Bacillus cell including, but notlimited to, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillusbrevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans,Bacillus firmus, Geobacillus stearothermophilus, Bacillus lautus,Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacilluspumilus, Bacillus stearothermophilus, Bacillus subtilis, and Bacillusthuringiensis cells.

The bacterial host cell may also be any Streptococcus cell including,but not limited to, Streptococcus equisimilis, Streptococcus pyogenes,Streptococcus uberis, and Streptococcus equi subsp. Zooepidemicus cells.

The bacterial host cell may also be any Streptomyces cell including, butnot limited to, Streptomyces achromogenes, Streptomyces avermitilis,Streptomyces coelicolor, Streptomyces griseus, and Streptomyces lividanscells.

The introduction of DNA into a Bacillus cell may, for instance, beeffected by protoplast transformation (see, e.g., Chang and Cohen, 1979,Mol. Gen. Genet. 168: 111-115), by using competent cells (see, e.g.,Young and Spizizen, 1961, J. Bacteriol. 81: 823-829, or Dubnau andDavidoff-Abelson, 1971, J. Mol. Biol. 56: 209-221), by electroporation(see, e.g., Shigekawa and Dower, 1988, Biotechniques 6: 742-751), or byconjugation (see, e.g., Koehler and Thorne, 1987, J. Bacteriol. 169:5271-5278). The introduction of DNA into an E. coli cell may, forinstance, be effected by protoplast transformation (see, e.g., Hanahan,1983, J. Mol. Biol. 166: 557-580) or electroporation (see, e.g., Doweret al., 1988, Nucleic Acids Res. 16: 6127-6145). The introduction of DNAinto a Streptomyces cell may, for instance, be effected by protoplasttransformation and electroporation (see, e.g., Gong et al., 2004, FoliaMicrobiol. (Praha) 49: 399-405), by conjugation (see, e.g., Mazodier etal., 1989, J. Bacteriol. 171: 3583-3585), or by transduction (see, e.g.,Burke et al., 2001, Proc. Natl. Acad. Sci. USA 98: 6289-6294). Theintroduction of DNA into a Pseudomonas cell may, for instance, beeffected by electroporation (see, e.g., Choi et al., 2006, J. Microbiol.Methods 64: 391-397) or by conjugation (see, e.g., Pinedo and Smets,2005, Appl. Environ. Microbiol. 71: 51-57). The introduction of DNA intoa Streptococcus cell may, for instance, be effected by naturalcompetence (see, e.g., Perry and Kuramitsu, 1981, Infect. Immun. 32:1295-1297), by protoplast transformation (see, e.g., Catt and Jollick,1991, Microbios 68: 189-207), by electroporation (see, e.g., Buckley etal., 1999, Appl. Environ. Microbiol. 65: 3800-3804) or by conjugation(see, e.g., Clewell, 1981, Microbiol. Rev. 45: 409-436). However, anymethod known in the art for introducing DNA into a host cell can beused.

The host cell may also be a eukaryote, such as a mammalian, insect,plant, or fungal cell.

The host cell may be a fungal cell. “Fungi” as used herein includes thephyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota (asdefined by Hawksworth et al., In, Ainsworth and Bisby's Dictionary ofThe Fungi, 8th edition, 1995, CAB International, University Press,Cambridge, UK) as well as the Oomycota (as cited in Hawksworth et al.,1995, supra, page 171) and all mitosporic fungi (Hawksworth et al.,1995, supra).

The fungal host cell may be a yeast cell. “Yeast” as used hereinincludes ascosporogenous yeast (Endomycetales), basidiosporogenousyeast, and yeast belonging to the Fungi Imperfecti (Blastomycetes).Since the classification of yeast may change in the future, for thepurposes of this invention, yeast shall be defined as described inBiology and Activities of Yeast (Skinner, F. A., Passmore, S. M., andDavenport, R. R., eds, Soc. App. Bacteriol. Symposium Series No. 9,1980).

The yeast host cell may be a Candida, Hansenula, Kluyveromyces, Pichia,Saccharomyces, Schizosaccharomyces, or Yarrowia cell such as aKluyveromyces lactis, Saccharomyces carlsbergensis, Saccharomycescerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii,Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomycesoviformis, or Yarrowia lipolytica cell.

The fungal host cell may be a filamentous fungal cell. “Filamentousfungi” include all filamentous forms of the subdivision Eumycota andOomycota (as defined by Hawksworth et al., 1995, supra). The filamentousfungi are generally characterized by a mycelial wall composed of chitin,cellulose, glucan, chitosan, mannan, and other complex polysaccharides.Vegetative growth is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such asSaccharomyces cerevisiae is by budding of a unicellular thallus andcarbon catabolism may be fermentative.

The filamentous fungal host cell may be an Acremonium, Aspergillus,Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus,Coriolus, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe,Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces,Penicillium, Phanerochaete, Phlebia, Piromyces, Pleurotus,Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium,Trametes, or Trichoderma cell.

For example, the filamentous fungal host cell may be an Aspergillusawamori, Aspergillus foetidus, Aspergillus fumigatus, Aspergillusjaponicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae,Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis caregiea,Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsisrivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora,Chrysosporium inops, Chrysosporium keratinophilum, Chrysosporiumlucknowense, Chrysosporium merdarium, Chrysosporium pannicola,Chrysosporium queenslandicum, Chrysosporium tropicum, Chrysosporiumzonatum, Coprinus cinereus, Coriolus hirsutus, Fusarium bactridioides,Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusariumgraminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi,Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusariumsambucinum, Fusarium sarcochroum, Fusarium sporotrichioides, Fusariumsuiphureum, Fusarium torulosum, Fusarium trichothecioides, Fusariumvenenatum, Humicola insolens, Humicola lanuginose, Mucor miehei,Myceliophthora thermophila, Neurospora crassa, Penicillium purpurogenum,Phanerochaete chrysosporium, Phiebia radiata, Pleurotus eryngii,Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichodermaharzianum, Trichoderma koningii, Trichoderma longibrachiatum,Trichoderma reesei, or Trichoderma viride cell. A specifically preferredhost cell is an Aspergillus oryzae cell.

Fungal cells may be transformed by a process involving protoplastformation, transformation of the protoplasts, and regeneration of thecell wall in a manner known per se. Suitable procedures fortransformation of Aspergillus and Trichoderma host cells are describedin EP 238023 and Yelton et al., 1984, Proc. Natl. Acad. Sci. USA 81:1470-1474. Suitable methods for transforming Fusarium species aredescribed by Malardier et al., 1989, Gene 78: 147-156, and WO 96/00787.Yeast may be transformed using the procedures described by Becker andGuarente, In Abelson, J. N. and Simon, M. I., editors, Guide to YeastGenetics and Molecular Biology, Methods in Enzymology, Volume 194, pp182-187, Academic Press, Inc., New York; Ito et al., 1983, J. Bacteriol.153: 163; and Hinnen et al., 1978, Proc. Natl. Acad. Sci. USA 75: 1920.

Methods of Production

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a cell, which inits wild-type form produces the polypeptide, under conditions conducivefor production of the polypeptide; and (b) recovering the polypeptide.In a preferred aspect, the cell is of the genus Bacillus. In a morepreferred aspect, the cell is Bacillus sp. 19138.

The present invention also relates to methods of producing a polypeptideof the present invention, comprising: (a) cultivating a recombinant hostcell of the present invention under conditions conducive for productionof the polypeptide; and (b) recovering the polypeptide.

The host cells are cultivated in a nutrient medium suitable forproduction of the polypeptide using methods well known in the art. Forexample, the cell may be cultivated by shake flask cultivation, andsmall-scale or large-scale fermentation (including continuous, batch,fed-batch, or solid state fermentations) in laboratory or industrialfermentors performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium comprising carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are available from commercial suppliers or may be preparedaccording to published compositions (e.g., in catalogues of the AmericanType Culture Collection). If the polypeptide is secreted into thenutrient medium, the polypeptide can be recovered directly from themedium. If the polypeptide is not secreted, it can be recovered fromcell lysates.

More details are provided in the Section on “Nucleic Acid Constructs,Expression Vectors, Recombinant Host Cells, and Methods for Productionof Proteases” below.

The polypeptide may be detected using methods known in the art that arespecific for the polypeptides. These detection methods may include useof specific antibodies, formation of an enzyme product, or disappearanceof an enzyme substrate. For example, an enzyme assay may be used todetermine the activity of the polypeptide.

The polypeptide may be recovered using methods known in the art. Forexample, the polypeptide may be recovered from the nutrient medium byconventional procedures including, but not limited to, centrifugation,filtration, extraction, spray-drying, evaporation, or precipitation.

The polypeptide may be purified by a variety of procedures known in theart including, but not limited to, chromatography (e.g., ion exchange,affinity, hydrophobic, chromatofocusing, and size exclusion),electrophoretic procedures (e.g., preparative isoelectric focusing),differential solubility (e.g., ammonium sulfate precipitation),SDS-PAGE, or extraction (see, e.g., Protein Purification, J.-C. Jansonand Lars Ryden, editors, VCH Publishers, New York, 1989) to obtainsubstantially pure polypeptides.

In an alternative aspect, the polypeptide is not recovered, but rather ahost cell of the present invention expressing a polypeptide is used as asource of the polypeptide.

Plants

The present invention also relates to plants, e.g., a transgenic plant,plant part, or plant cell, comprising an isolated polynucleotide of thepresent invention so as to express and produce the polypeptide inrecoverable quantities. The polypeptide may be recovered from the plantor plant part. Alternatively, the plant or plant part containing thepolypeptide may be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

The transgenic plant can be dicotyledonous (a dicot) or monocotyledonous(a monocot). Examples of monocot plants are grasses, such as meadowgrass (blue grass, Poa), forage grass such as Festuca, Lolium, temperategrass, such as Agrostis, and cereals, e.g., wheat, oats, rye, barley,rice, sorghum, and maize (corn).

Examples of dicot plants are tobacco, legumes, such as lupins, potato,sugar beet, pea, bean and soybean, and cruciferous plants (familyBrassicaceae), such as cauliflower, rape seed, and the closely relatedmodel organism Arabidopsis thaliana.

Examples of plant parts are stem, callus, leaves, root, fruits, seeds,and tubers as well as the individual tissues comprising these parts,e.g., epidermis, mesophyll, parenchyme, vascular tissues, meristems.Specific plant cell compartments, such as chloroplasts, apoplasts,mitochondria, vacuoles, peroxisomes and cytoplasm are also considered tobe a plant part. Furthermore, any plant cell, whatever the tissueorigin, is considered to be a plant part. Likewise, plant parts such asspecific tissues and cells isolated to facilitate the utilization of theinvention are also considered plant parts, e.g., embryos, endosperms,aleurone and seeds coats.

Also included within the scope of the present invention are the progenyof such plants, plant parts, and plant cells.

The transgenic plant or plant cell expressing a polypeptide may beconstructed in accordance with methods known in the art. In short, theplant or plant cell is constructed by incorporating one or more(several) expression constructs encoding a polypeptide into the planthost genome or chloroplast genome and propagating the resulting modifiedplant or plant cell into a transgenic plant or plant cell.

The expression construct is conveniently a nucleic acid construct thatcomprises a polynucleotide encoding a polypeptide operably linked withappropriate regulatory sequences required for expression of thepolynucleotide in the plant or plant part of choice. Furthermore, theexpression construct may comprise a selectable marker useful foridentifying host cells into which the expression construct has beenintegrated and DNA sequences necessary for introduction of the constructinto the plant in question (the latter depends on the DNA introductionmethod to be used).

The choice of regulatory sequences, such as promoter and terminatorsequences and optionally signal or transit sequences, is determined, forexample, on the basis of when, where, and how the polypeptide is desiredto be expressed. For instance, the expression of the gene encoding apolypeptide may be constitutive or inducible, or may be developmental,stage or tissue specific, and the gene product may be targeted to aspecific tissue or plant part such as seeds or leaves. Regulatorysequences are, for example, described by Tague et al., 1988, PlantPhysiology 86: 506.

For constitutive expression, the 35S-CaMV, the maize ubiquitin 1, andthe rice actin 1 promoter may be used (Franck et al., 1980, Cell 21:285-294; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689; Zhanget al., 1991, Plant Cell 3: 1155-1165). Organ-specific promoters may be,for example, a promoter from storage sink tissues such as seeds, potatotubers, and fruits (Edwards and Coruzzi, 1990, Ann. Rev. Genet. 24:275-303), or from metabolic sink tissues such as meristems (Ito et al.,1994, Plant Mol. Biol. 24: 863-878), a seed specific promoter such asthe glutelin, prolamin, globulin, or albumin promoter from rice (Wu etal., 1998, Plant Cell Physiol. 39: 885-889), a Vicia faba promoter fromthe legumin B4 and the unknown seed protein gene from Vicia faba (Conradet al., 1998, J. Plant Physiol. 152: 708-711), a promoter from a seedoil body protein (Chen et al., 1998, Plant Cell Physiol. 39: 935-941),the storage protein napA promoter from Brassica napus, or any other seedspecific promoter known in the art, e.g., as described in WO 91/14772.Furthermore, the promoter may be a leaf specific promoter such as therbcs promoter from rice or tomato (Kyozuka et al., 1993, Plant Physiol.102: 991-1000), the chlorella virus adenine methyltransferase genepromoter (Mitra and Higgins, 1994, Plant Mol. Biol. 26: 85-93), the aldPgene promoter from rice (Kagaya et al., 1995, Mol. Gen. Genet. 248:668-674), or a wound inducible promoter such as the potato pin2 promoter(Xu et al., 1993, Plant Mol. Biol. 22: 573-588). Likewise, the promotermay be inducible by abiotic treatments such as temperature, drought, oralterations in salinity or induced by exogenously applied substancesthat activate the promoter, e.g., ethanol, oestrogens, plant hormonessuch as ethylene, abscisic acid, and gibberellic acid, and heavy metals.

A promoter enhancer element may also be used to achieve higherexpression of a polypeptide in the plant. For instance, the promoterenhancer element may be an intron that is placed between the promoterand the polynucleotide encoding a polypeptide. For instance, Xu et al.,1993, supra, disclose the use of the first intron of the rice actin 1gene to enhance expression.

The selectable marker gene and any other parts of the expressionconstruct may be chosen from those available in the art.

The nucleic acid construct is incorporated into the plant genomeaccording to conventional techniques known in the art, includingAgrobacterium-mediated transformation, virus-mediated transformation,microinjection, particle bombardment, biolistic transformation, andelectroporation (Gasser et al., 1990, Science 244: 1293; Potrykus, 1990,Bio/Technology 8: 535; Shimamoto et al., 1989, Nature 338: 274).

Presently, Agrobacterium tumefaciens-mediated gene transfer is themethod of choice for generating transgenic dicots (for a review, seeHooykas and Schilperoort, 1992, Plant Mol. Biol. 19: 15-38) and can alsobe used for transforming monocots, although other transformation methodsare often used for these plants. Presently, the method of choice forgenerating transgenic monocots is particle bombardment (microscopic goldor tungsten particles coated with the transforming DNA) of embryoniccalli or developing embryos (Christou, 1992, Plant J. 2: 275-281;Shimamoto, 1994, Curr. Opin. Biotechnol. 5: 158-162; Vasil et al., 1992,Bio/Technology 10: 667-674). An alternative method for transformation ofmonocots is based on protoplast transformation as described by Omirullehet al., 1993, Plant Mol. Biol. 21: 415-428. Additional transformationmethods for use in accordance with the present disclosure include thosedescribed in U.S. Pat. Nos. 6,395,966 and 7,151,204 (both of which areherein incorporated by reference in their entirety).

Following transformation, the transformants having incorporated theexpression construct are selected and regenerated into whole plantsaccording to methods well known in the art. Often the transformationprocedure is designed for the selective elimination of selection geneseither during regeneration or in the following generations by using, forexample, co-transformation with two separate T-DNA constructs orsite-specific excision of the selection gene by a specific recombinase.

In addition to direct transformation of a particular plant genotype witha construct prepared according to the present invention, transgenicplants may be made by crossing a plant having the construct to a secondplant lacking the construct. For example, a construct encoding apolypeptide can be introduced into a particular plant variety bycrossing, without the need for ever directly transforming a plant ofthat given variety. Therefore, the present invention encompasses notonly a plant directly regenerated from cells which have been transformedin accordance with the present invention, but also the progeny of suchplants. As used herein, progeny may refer to the offspring of anygeneration of a parent plant prepared in accordance with the presentinvention. Such progeny may include a DNA construct prepared inaccordance with the present invention, or a portion of a DNA constructprepared in accordance with the present invention. Crossing results inthe introduction of a transgene into a plant line by cross pollinating astarting line with a donor plant line. Non-limiting examples of suchsteps are further articulated in U.S. Pat. No. 7,151,204.

Plants may be generated through a process of backcross conversion. Forexample, plants include plants referred to as a backcross convertedgenotype, line, inbred, or hybrid.

Genetic markers may be used to assist in the introgression of one ormore transgenes of the invention from one genetic background intoanother. Marker assisted selection offers advantages relative toconventional breeding in that it can be used to avoid errors caused byphenotypic variations. Further, genetic markers may provide dataregarding the relative degree of elite germplasm in the individualprogeny of a particular cross. For example, when a plant with a desiredtrait which otherwise has a non-agronomically desirable geneticbackground is crossed to an elite parent, genetic markers may be used toselect progeny which not only possess the trait of interest, but alsohave a relatively large proportion of the desired germplasm. In thisway, the number of generations required to introgress one or more traitsinto a particular genetic background is minimized.

The present invention also relates to methods of producing a polypeptideof the present invention comprising: (a) cultivating a transgenic plantor a plant cell comprising a polynucleotide encoding the polypeptideunder conditions conducive for production of the polypeptide; and (b)recovering the polypeptide.

Compositions

The present invention also relates to compositions comprising a proteaseof the present invention. Preferably, the compositions are enriched insuch a protease. The term “enriched” indicates that the proteaseactivity of the composition has been increased, e.g., with an enrichmentfactor of at least 1.1.

In one aspect, the composition comprises an isolated polypeptide havingprotease activity, selected from the group consisting of:

(a) a polypeptide having at least 60% sequence identity to thepolypeptide of SEQ ID NO: 5, SEQ ID NO: 6, or the mature polypeptide ofSEQ ID NO: 2 or SEQ ID NO: 4;

(b) a polypeptide having at least 60% sequence identity to thepolypeptide of SEQ ID NO: 19, or the mature polypeptide of SEQ ID NO: 16or SEQ ID NO: 18;

(c) a polypeptide having at least 60% sequence identity to thepolypeptide of SEQ ID NO: 24, or the mature polypeptide of SEQ ID NO: 21or SEQ ID NO: 23;

(d) a polypeptide encoded by a polynucleotide that hybridizes undermedium stringency conditions, medium-high stringency conditions, highstringency conditions, or very high stringency conditions with

-   -   (i) the mature polypeptide coding sequence of SEQ ID NO: 1,    -   (ii) the mature polypeptide coding sequence of SEQ ID NO: 3,    -   (iii) the mature polypeptide coding sequence of SEQ ID NO: 15,    -   (iv) the mature polypeptide coding sequence of SEQ ID NO: 17,    -   (v) the mature polypeptide coding sequence of SEQ ID NO: 20,    -   (vi) the mature polypeptide coding sequence of SEQ ID NO: 22,    -   (vii) the full-length complementary strand of (i), (ii), (iii),        (iv), (v) or (vi);

(e) a polypeptide encoded by a polynucleotide having at least 84%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 1 or SEQ ID NO: 3;

(f) a polypeptide encoded by a polynucleotide having at least 83%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 15 or SEQ ID NO: 17;

(g) a polypeptide encoded by a polynucleotide having at least 85%sequence identity to the mature polypeptide coding sequence of SEQ IDNO: 20 or SEQ ID NO: 22;

(h) a variant of the polypeptide of SEQ ID NO: 5, SEQ ID NO: 6, SEQ IDNO: 19 or SEQ ID NO: 24, or the mature polypeptide of SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 21 or SEQ ID NO: 23comprising a substitution, deletion, and/or insertion at one or more(several) positions; and

(i) a fragment of a polypeptide of (a), (b), (c), (d), (e), (f), (g) or(h) having protease activity.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 70% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 75% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 80% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 85% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 87% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 90% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 91% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 92% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 93% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 94% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 95% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 96% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 97% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 98% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 99% sequence identity to the polypeptide of SEQ ID NO:5, SEQ ID NO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO:4.

An embodiment of the invention is a composition comprising a polypeptidehaving 100% sequence identity to the polypeptide of SEQ ID NO: 5, SEQ IDNO: 6, or the mature polypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

In one aspect, the composition comprises or consists of the amino acidsequence of SEQ ID NO: 5, SEQ ID NO: 6, or the mature polypeptide of SEQID NO: 2 or SEQ ID NO: 4 or an allelic variant thereof; or is a fragmentthereof having protease activity. In another aspect, the compositioncomprises or consists of the mature polypeptide of SEQ ID NO: 2 or SEQID NO: 4. In a further aspect, the composition comprises or consists ofthe polypeptide of SEQ ID NO: 5 or SEQ ID NO: 6. In another aspect, thecomposition comprises or consists of amino acids 1 to 366 of SEQ ID NO:2, amino acids 1 to 366 of SEQ ID NO: 4, amino acids 1 to 366 of SEQ IDNO: 5 or amino acids 1 to 366 of SEQ ID NO: 6.

In an embodiment, the variant comprising a substitution, deletion,and/or insertion of one or more (several) amino acids of SEQ ID NO: 3has at least 60%, e.g., at least 70%, at least 75%, at least 80%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, but less than 100%sequence identity to SEQ ID NO: 5, SEQ ID NO: 6, or the maturepolypeptide of SEQ ID NO: 2 or SEQ ID NO: 4.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 70% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 75% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 80% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 85% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 87% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 90% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 91% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 92% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 93% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 94% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 95% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 96% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 97% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 98% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 99% sequence identity to the polypeptide of SEQ ID NO:19, or the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a composition comprising a polypeptidehaving 100% sequence identity to the polypeptide of SEQ ID NO: 19, orthe mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.

In one aspect, the composition comprises or consists of the amino acidsequence of SEQ ID NO: 19, or the mature polypeptide of SEQ ID NO: 16 orSEQ ID NO: 18 or an allelic variant thereof; or is a fragment thereofhaving protease activity. In another aspect, the composition comprisesor consists of the mature polypeptide of SEQ ID NO: 16 or SEQ ID NO: 18.In a further aspect, the composition comprises or consists of thepolypeptide of SEQ ID NO: 19. In another aspect, the compositioncomprises or consists of amino acids 1 to 366 of SEQ ID NO: 16, aminoacids 1 to 366 of SEQ ID NO: 18, or amino acids 1 to 366 of SEQ ID NO:19.

In an embodiment, the variant comprising a substitution, deletion,and/or insertion of one or more (several) amino acids of SEQ ID NO: 19has at least 60%, e.g., at least 70%, at least 75%, at least 80%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, but less than 100%sequence identity to SEQ ID NO: 19, or the mature polypeptide of SEQ IDNO: 16 or SEQ ID NO: 18.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 70% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 75% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 80% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 85% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 87% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 90% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 91% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 92% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 93% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 94% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 95% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 96% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 97% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 98% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a composition comprising a polypeptidehaving at least 99% sequence identity to the polypeptide of SEQ ID NO:24, or the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

An embodiment of the invention is a composition comprising a polypeptidehaving 100% sequence identity to the polypeptide of SEQ ID NO: 24, orthe mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.

In one aspect, the composition comprises or consists of the amino acidsequence of SEQ ID NO: 24, or the mature polypeptide of SEQ ID NO: 21 orSEQ ID NO: 23 or an allelic variant thereof; or is a fragment thereofhaving protease activity. In another aspect, the composition comprisesor consists of the mature polypeptide of SEQ ID NO: 21 or SEQ ID NO: 23.In a further aspect, the composition comprises or consists of thepolypeptide of SEQ ID NO: 24. In another aspect, the compositioncomprises or consists of amino acids 1 to 366 of SEQ ID NO: 21, aminoacids 1 to 366 of SEQ ID NO: 23, or amino acids 1 to 366 of SEQ ID NO:24.

In an embodiment, the variant comprising a substitution, deletion,and/or insertion of one or more (several) amino acids of SEQ ID NO: 24has at least 60%, e.g., at least 70%, at least 75%, at least 80%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99%, but less than 100%sequence identity to SEQ ID NO: 24, or the mature polypeptide of SEQ IDNO: 21 or SEQ ID NO: 23

In a preferred embodiment, the composition is an animal feed compositionwhich further comprises one or more amylases, phytases, xylanases,galactanases, alpha-galactosidases, proteases, phospholipases,beta-glucanases, or any mixture thereof.

In another preferred embodiment, the composition is an animal feedadditive which further comprises at least one fat-soluble vitamin,and/or at least one water-soluble vitamin, and/or at least one tracemineral. The animal feed additive may further comprise one or moreamylases, phytases, xylanases, galactanases, alpha-galactosidases,proteases, phospholipases, beta-glucanases, or any mixture thereof.

The composition may comprise a protease of the present invention as themajor enzymatic component, e.g., a mono-component composition.Alternatively, the composition may comprise multiple enzymaticactivities, such as an aminopeptidase, amylase, carbohydrase,carboxypeptidase, catalase, cellulase, chitinase, cutinase, cyclodextringlycosyltransferase, deoxyribonuclease, esterase, alpha-galactosidase,beta-galactosidase, glucoamylase, alpha-glucosidase, beta-glucosidase,haloperoxidase, invertase, laccase, lipase, mannosidase, oxidase,pectinolytic enzyme, peptidoglutaminase, peroxidase, phytase,polyphenoloxidase, proteolytic enzyme, ribonuclease, transglutaminase,or xylanase. The additional enzyme(s) may be produced, for example, bymicroorganisms such as bacteria or fungi or by plants or by animals. Thecompositions may be prepared in accordance with methods known in the artand may be in the form of a liquid or a dry composition. For instance,the composition may be in the form of a granulate or a microgranulate.The protease may be stabilized in accordance with methods known in theart.

Uses

The present invention is also directed to methods for using thepolypeptides having protease activity, or compositions thereof, for,e.g., animal feed.

Use in Animal Feed

The present invention is also directed to methods for using theproteases having protease activity in animal feed, as well as to feedcompositions and feed additives comprising the proteases of theinvention.

The term animal includes all animals. Examples of animals arenon-ruminants, and ruminants. Ruminant animals include, for example,animals such as sheep, goats, and cattle, e.g., beef cattle, cows, andyoung calves. In a particular embodiment, the animal is a non-ruminantanimal. Non-ruminant animals include mono-gastric animals, e.g., pigs orswine (including, but not limited to, piglets, growing pigs, and sows);poultry such as turkeys, ducks and chicken (including but not limited tobroiler chicks, layers); horses (including but not limited to hotbloods,coldbloods and warm bloods), young calves; and fish (including but notlimited to salmon, trout, tilapia, catfish and carps; and crustaceans(including but not limited to shrimps and prawns).

The term feed or feed composition means any compound, preparation,mixture, or composition suitable for, or intended for intake by ananimal.

In the use according to the invention the protease can be fed to theanimal before, after, or simultaneously with the diet. The latter ispreferred.

In a particular embodiment, the protease, in the form in which it isadded to the feed, or when being included in a feed additive, iswell-defined. Well-defined means that the protease preparation is atleast 50% pure as determined by Size-exclusion chromatography (seeExample 12 of WO 01/58275). In other particular embodiments, theprotease preparation is at least 60, 70, 80, 85, 88, 90, 92, 94, or atleast 95% pure as determined by this method.

A well-defined protease preparation is advantageous. For instance, it ismuch easier to dose correctly to the feed a protease that is essentiallyfree from interfering or contaminating other proteases. The term dosecorrectly refers in particular to the objective of obtaining consistentand constant results, and the capability of optimising dosage based uponthe desired effect.

For the use in animal feed, however, the protease need not be that pure;it may, e.g., include other enzymes, in which case it could be termed aprotease preparation.

The protease preparation can be (a) added directly to the feed (or useddirectly in a protein treatment process), or (b) it can be used in theproduction of one or more intermediate compositions such as feedadditives or premixes that is subsequently added to the feed (or used ina treatment process). The degree of purity described above refers to thepurity of the original protease preparation, whether used according to(a) or (b) above.

Protease preparations with purities of this order of magnitude are inparticular obtainable using recombinant methods of production, whereasthey are not so easily obtained and also subject to a much higherbatch-to-batch variation when the protease is produced by traditionalfermentation methods.

Such protease preparation may of course be mixed with other enzymes.

The protein may be an animal protein, such as meat and bone meal,feather meal, and/or fish meal; or it may be a vegetable protein.

The term vegetable proteins as used herein refers to any compound,composition, preparation or mixture that includes at least one proteinderived from or originating from a vegetable, including modifiedproteins and protein-derivatives. In particular embodiments, the proteincontent of the vegetable proteins is at least 10, 20, 30, 40, 50, or 60%(w/w).

Vegetable proteins may be derived from vegetable protein sources, suchas legumes and cereals, for example materials from plants of thefamilies Fabaceae (Leguminosae), Cruciferaceae, Chenopodiaceae, andPoaceae, such as soy bean meal, lupin meal and rapeseed meal.

In a particular embodiment, the vegetable protein source is materialfrom one or more plants of the family Fabaceae, e.g., soybean, lupine,pea, or bean.

In another particular embodiment, the vegetable protein source ismaterial from one or more plants of the family Chenopodiaceae, e.g.,beet, sugar beet, spinach or quinoa.

Other examples of vegetable protein sources are rapeseed, sunflowerseed, cotton seed, and cabbage.

Soybean is a preferred vegetable protein source.

Other examples of vegetable protein sources are cereals such as barley,wheat, rye, oat, maize (corn), rice, triticale, and sorghum.

In a particular embodiment of a treatment process, the protease(s) inquestion is affecting (or acting on, or exerting its hydrolyzing ordegrading influence on) the proteins, such as vegetable proteins orprotein sources. To achieve this, the protein or protein source istypically suspended in a solvent, eg an aqueous solvent such as water,and the pH and temperature values are adjusted paying due regard to thecharacteristics of the enzyme in question. For example, the treatmentmay take place at a pH-value at which the activity of the actualprotease is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or atleast 90%. Likewise, for example, the treatment may take place at atemperature at which the activity of the actual protease is at least 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or at least 90%. The abovepercentage activity indications are relative to the maximum activities.The enzymatic reaction is continued until the desired result isachieved, following which it may or may not be stopped by inactivatingthe enzyme, e.g., by a heat-treatment step.

In another particular embodiment of a treatment process of theinvention, the protease action is sustained, meaning, e.g., that theprotease is added to the proteins, but its hydrolyzing influence is soto speak not switched on until later when desired, once suitablehydrolyzing conditions are established, or once any enzyme inhibitorsare inactivated, or whatever other means could have been applied topostpone the action of the enzyme.

In one embodiment, the treatment is a pre-treatment of animal feed orproteins for use in animal feed, i.e., the proteins are hydrolyzedbefore intake.

The term improving the nutritional value of an animal feed meansimproving the availability of nutrients in the feed. In this inventionimproving the nutritional values refers in particular to improving theavailability of the protein fraction of the feed, thereby leading toincreased protein extraction, higher protein yields, and/or improvedprotein utilization. When the nutritional value of the feed isincreased, the protein and/or amino acid digestibility is increased andthe growth rate and/or weight gain and/or feed conversion (i.e., theweight of ingested feed relative to weight gain) of the animal might beimproved.

The protease can be added to the feed in any form, be it as a relativelypure protease or in admixture with other components intended foraddition to animal feed, i.e., in the form of animal feed additives,such as the so-called pre-mixes for animal feed.

In a further aspect, the present invention relates to compositions foruse in animal feed, such as animal feed, and animal feed additives,e.g., premixes.

Apart from the protease of the invention, the animal feed additives ofthe invention contain at least one fat-soluble vitamin, and/or at leastone water soluble vitamin, and/or at least one trace mineral, and/or atleast one macro mineral.

Further, optional, feed-additive ingredients are colouring agents, e.g.,carotenoids such as beta-carotene, astaxanthin, and lutein; stabilisers;growth improving additives and aroma compounds/flavorings, e.g.,creosol, anethol, deca-, undeca- and/or dodeca-lactones, ionones, irone,gingerol, piperidine, propylidene phatalide, butylidene phatalide,capsaicin and/or tannin; antimicrobial peptides; polyunsaturated fattyacids (PUFAs); reactive oxygen generating species; also, a support maybe used that may contain, for example, 40-50% by weight of wood fibres,8-10% by weight of stearine, 4-5% by weight of curcuma powder, 4-58% byweight of rosemary powder, 22-28% by weight of limestone, 1-3% by weightof a gum, such as gum arabic, 5-50% by weight of sugar and/or starch and5-15% by weight of water.

A feed or a feed additive of the invention may also comprise at leastone other enzyme selected from amongst phytase (EC 3.1.3.8 or 3.1.3.26);xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89); alpha-galactosidase(EC 3.2.1.22); further protease (EC 3.4); phospholipase A1 (EC3.1.1.32); phospholipase A2 (EC 3.1.1.4); lysophospholipase (EC3.1.1.5); phospholipase C (3.1.4.3); phospholipase D (EC 3.1.4.4);amylase such as, for example, alpha-amylase (EC 3.2.1.1); lysozyme (EC3.2.1.17); and/or beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6).

In a particular embodiment, the feed or a feed additive of the inventionalso comprises a phytase (EC 3.1.3.8 or 3.1.3.26).

In a particular embodiment, the feed or a feed additive of the inventionalso comprises a xylanase (EC 3.2.1.8).

A feed or a feed additive of the invention may also comprise at leastone probiotic or direct fed microbial (DFM) optionally together with oneor more other enzymes selected from amongst phytase (EC 3.1.3.8 or3.1.3.26); xylanase (EC 3.2.1.8); galactanase (EC 3.2.1.89);alpha-galactosidase (EC 3.2.1.22); further protease (EC 3.4),phospholipase A1 (EC 3.1.1.32); phospholipase A2 (EC 3.1.1.4);lysophospholipase (EC 3.1.1.5); phospholipase C (3.1.4.3); phospholipaseD (EC 3.1.4.4); amylase such as, for example, alpha-amylase (EC3.2.1.1); and/or beta-glucanase (EC 3.2.1.4 or EC 3.2.1.6).

The direct fed microbial may be a bacterium from one or more of thefollowing genera: Lactobacillus, Lactococcus, Streptococcus, Bacillus,Pediococcus, Enterococcus, Leuconostoc, Carnobacterium,Propionibacterium, Bifidobacterium, Clostridium and Megasphaera or anycombination thereof, preferably from Bacillus subtilis, Bacilluslicheniformis, Bacillus amyloliquefaciens, Enterococcus faecium,Enterococcus spp, and Pediococcus spp, Lactobacillus spp,Bifidobacterium spp, Lactobacillus acidophilus, Pediococsusacidilactici, Lactococcus lactis, Bifidobacterium bifidum,Propionibacterium thoenii, Lactobacillus farciminus, lactobacillusrhamnosus, Clostridium butyricum, Bifidobacterium animalis ssp.animalis, Lactobacillus reuteri, Bacillus cereus, Lactobacillussalivarius ssp. salivarius, Megasphaera elsdenii, Propionibacteria spand more preferably from Bacillus subtilis strains 3A-P4 (PTA-6506);15A-P4 (PTA-6507); 22C-P1 (PTA-6508); 2084 (NRRL B-500130); LSSA01(NRRL-B-50104); BS27 (NRRL B-501 05); BS 18 (NRRL B-50633); and BS 278(NRRL B-50634).

In a particular embodiment, these other enzymes are well-defined (asdefined above for protease preparations).

Examples of antimicrobial peptides (AMP's) are CAP18, Leucocin A,Tritrpticin, Protegrin-1, Thanatin, Defensin, Lactoferrin,Lactoferricin, and Ovispirin such as Novispirin (Robert Lehrer, 2000),Plectasins, and Statins, including the compounds and polypeptidesdisclosed in WO 03/044049 and WO 03/048148, as well as variants orfragments of the above that retain antimicrobial activity.

Examples of antifungal polypeptides (AFP's) are the Aspergillusgiganteus, and Aspergillus niger peptides, as well as variants andfragments thereof which retain antifungal activity, as disclosed in WO94/01459 and WO 02/090384.

Examples of polyunsaturated fatty acids are C18, C20 and C22polyunsaturated fatty acids, such as arachidonic acid, docosohexaenoicacid, eicosapentaenoic acid and gamma-linoleic acid.

Examples of reactive oxygen generating species are chemicals such asperborate, persulphate, or percarbonate; and enzymes such as an oxidase,an oxygenase or a syntethase.

Usually fat- and water-soluble vitamins, as well as trace minerals formpart of a so-called premix intended for addition to the feed, whereasmacro minerals are usually separately added to the feed. Either of thesecomposition types, when enriched with a protease of the invention, is ananimal feed additive of the invention.

In a particular embodiment, the animal feed additive of the invention isintended for being included (or prescribed as having to be included) inanimal diets or feed at levels of 0.01 to 10.0%; more particularly 0.05to 5.0%; or 0.2 to 1.0% (% meaning g additive per 100 g feed). This isso in particular for premixes.

The following are non-exclusive lists of examples of these components:

Examples of fat-soluble vitamins are vitamin A, vitamin D3, vitamin E,and vitamin K, e.g., vitamin K3.

Examples of water-soluble vitamins are vitamin B12, biotin and choline,vitamin B1, vitamin B2, vitamin B6, niacin, folic acid andpanthothenate, e.g., Ca-D-panthothenate.

Examples of trace minerals are manganese, zinc, iron, copper, iodine,selenium, and cobalt.

Examples of macro minerals are calcium, phosphorus and sodium.

The nutritional requirements of these components (exemplified withpoultry and piglets/pigs) are listed in Table A of WO 01/58275.Nutritional requirement means that these components should be providedin the diet in the concentrations indicated.

In the alternative, the animal feed additive of the invention comprisesat least one of the individual components specified in Table A of WO01/58275. At least one means either of, one or more of, one, or two, orthree, or four and so forth up to all thirteen, or up to all fifteenindividual components. More specifically, this at least one individualcomponent is included in the additive of the invention in such an amountas to provide an in-feed-concentration within the range indicated incolumn four, or column five, or column six of Table A.

In a still further embodiment, the animal feed additive of the inventioncomprises at least one of the below vitamins, preferably to provide anin-feed-concentration within the ranges specified in the below Table 1(for piglet diets, and broiler diets, respectively).

TABLE 1 Typical vitamin recommendations Vitamin Piglet diet Broiler dietVitamin A 10,000-15,000 IU/kg feed 8-12,500 IU/kg feed Vitamin D31800-2000 IU/kg feed 3000-5000 IU/kg feed Vitamin E 60-100 mg/kg feed150-240 mg/kg feed Vitamin K3 2-4 mg/kg feed 2-4 mg/kg feed Vitamin B12-4 mg/kg feed 2-3 mg/kg feed Vitamin B2 6-10 mg/kg feed 7-9 mg/kg feedVitamin B6 4-8 mg/kg feed 3-6 mg/kg feed Vitamin B12 0.03-0.05 mg/kgfeed 0.015-0.04 mg/kg feed Niacin 30-50 mg/kg feed 50-80 mg/kg feed(Vitamin B3) Pantothenic 20-40 mg/kg feed 10-18 mg/kg feed acid Folicacid 1-2 mg/kg feed 1-2 mg/kg feed Biotin 0.15-0.4 mg/kg feed 0.15-0.3mg/kg feed Choline 200-400 mg/kg feed 300-600 mg/kg feed chloride

The present invention also relates to animal feed compositions. Animalfeed compositions or diets have a relatively high content of protein.Poultry and pig diets can be characterised as indicated in Table B of WO01/58275, columns 2-3. Fish diets can be characterised as indicated incolumn 4 of this Table B. Furthermore, such fish diets usually have acrude fat content of 200-310 g/kg.

WO 01/58275 corresponds to U.S. Ser. No. 09/779,334 which is herebyincorporated by reference.

An animal feed composition according to the invention has a crudeprotein content of 50-800 g/kg, and furthermore comprises at least oneprotease as claimed herein.

Furthermore, or in the alternative (to the crude protein contentindicated above), the animal feed composition of the invention has acontent of metabolisable energy of 10-30 MJ/kg; and/or a content ofcalcium of 0.1-200 g/kg; and/or a content of available phosphorus of0.1-200 g/kg; and/or a content of methionine of 0.1-100 g/kg; and/or acontent of methionine plus cysteine of 0.1-150 g/kg; and/or a content oflysine of 0.5-50 g/kg.

In particular embodiments, the content of metabolisable energy, crudeprotein, calcium, phosphorus, methionine, methionine plus cysteine,and/or lysine is within any one of ranges 2, 3, 4 or 5 in Table B of WO01/58275 (R. 2-5).

Crude protein is calculated as nitrogen (N) multiplied by a factor 6.25,i.e., Crude protein (g/kg)=N (g/kg)×6.25. The nitrogen content isdetermined by the Kjeldahl method (A.O.A.C., 1984, Official Methods ofAnalysis 14th ed., Association of Official Analytical Chemists,Washington D.C.).

Metabolisable energy can be calculated on the basis of the NRCpublication Nutrient requirements in swine, ninth revised edition 1988,subcommittee on swine nutrition, committee on animal nutrition, board ofagriculture, national research council. National Academy Press,Washington, D.C., pp. 2-6, and the European Table of Energy Values forPoultry Feed-stuffs, Spelderholt centre for poultry research andextension, 7361 DA Beekbergen, The Netherlands. Grafisch bedrijf Ponsen& looijen by, Wageningen. ISBN 90-71463-12-5.

The dietary content of calcium, available phosphorus and amino acids incomplete animal diets is calculated on the basis of feed tables such asVeevoedertabel 1997, gegevens over chemische samenstelling,verteerbaarheid en voederwaarde van voedermiddelen, CentralVeevoederbureau, Runderweg 6, 8219 pk Lelystad. ISBN 90-72839-13-7.

In a particular embodiment, the animal feed composition of the inventioncontains at least one vegetable protein as defined above.

The animal feed composition of the invention may also contain animalprotein, such as Meat and Bone Meal, Feather meal, and/or Fish Meal,typically in an amount of 0-25%. The animal feed composition of theinvention may also comprise Dried Destillers Grains with Solubles(DDGS), typically in amounts of 0-30%.

In still further particular embodiments, the animal feed composition ofthe invention contains 0-80% maize; and/or 0-80% sorghum; and/or 0-70%wheat; and/or 0-70% Barley; and/or 0-30% oats; and/or 0-40% soybeanmeal; and/or 0-25% fish meal; and/or 0-25% meat and bone meal; and/or0-20% whey.

Animal diets can, e.g., be manufactured as mash feed (non-pelleted) orpelleted feed. Typically, the milled feed-stuffs are mixed andsufficient amounts of essential vitamins and minerals are addedaccording to the specifications for the species in question. Enzymes canbe added as solid or liquid enzyme formulations. For example, for mashfeed a solid or liquid enzyme formulation may be added before or duringthe ingredient mixing step. For pelleted feed, the (liquid or solid)protease/enzyme preparation may also be added before or during the feedingredient step. Typically, a liquid protease/enzyme preparation isadded after the pelleting step. The enzyme may also be incorporated in afeed additive or premix.

The final enzyme concentration in the diet is within the range of0.01-200 mg enzyme protein per kg diet, for example in the range of0.5-25 mg enzyme protein per kg animal diet.

The protease should of course be applied in an effective amount, i.e.,in an amount adequate for improving hydrolysis, digestibility, and/orimproving nutritional value of feed. It is at present contemplated thatthe enzyme is administered in one or more of the following amounts(dosage ranges): 0.01-200; 0.01-100; 0.5-100; 1-50; 5-100; 10-100;0.05-50; or 0.10-10—all these ranges being in mg protease protein per kgfeed (ppm).

For determining mg protease protein per kg feed, the protease ispurified from the feed composition, and the specific activity of thepurified protease is determined using a relevant assay (see underprotease activity, substrates, and assays). The protease activity of thefeed composition as such is also determined using the same assay, and onthe basis of these two determinations, the dosage in mg protease proteinper kg feed is calculated.

The same principles apply for determining mg protease protein in feedadditives. Of course, if a sample is available of the protease used forpreparing the feed additive or the feed, the specific activity isdetermined from this sample (no need to purify the protease from thefeed composition or the additive).

Nucleic Acid Constructs, Expression Vectors, Recombinant Host Cells, andMethods for Production of Proteases

The present invention also relates to nucleic acid constructs,expression vectors and recombinant host cells comprising suchpolynucleotides encoding the proteases of the invention.

The present invention also relates to methods of producing a protease,comprising: (a) cultivating a recombinant host cell comprising suchpolynucleotide; and (b) recovering the protein.

The protein may be native or heterologous to a host cell. The term“protein” is not meant herein to refer to a specific length of theencoded product and, therefore, encompasses peptides, oligopeptides, andproteins. The term “protein” also encompasses two or more polypeptidescombined to form the encoded product. The proteins also include hybridpolypeptides and fused polypeptides.

Preferably, the protein is a protease. For example, the protein may be ahydrolase, such as a proteolytic enzyme or protease.

The gene may be obtained from any prokaryotic, eukaryotic, or othersource.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES Materials and Methods Media

DAP4C-1 medium was composed of 0.5 g yeast extract, 10 g maltose, 20 gdextrose, 11 g magnesium sulphate heptahydrate, 1 g dipotassiumphosphate, 2 g citric acid monohydrate, 5.2 g potassium phosphatetribasic monohydrate, 1 ml Dowfax 63N10 (antifoaming agent), 2.5 gcalcium carbonate, supplemented with 1 ml KU6 metal solution, anddeionised water to 1000 ml.

KU6 metal solution was composed of 6.8 g ZnCl₂, 2.5 g CuSO₄.5H₂O, 0.13 gNiCl₂, 13.9 g FeSO₄.7H₂O, 8.45 g MnSO₄.H₂O, 3 g C₆H₈O₇.H₂O, anddeionised water to 1000 ml.

LB plates were composed of 10 g of Bacto-tryptone, 5 g of yeast extract,10 g of sodium chloride, 15 g of Bacto-agar, and deionised water to 1000ml.

LB medium was composed of 10 g of Bacto-tryptone, 5 g of yeast extract,and 10 g of sodium chloride, and deionised water to 1000 ml.

COVE-Sucrose-T plates were composed of 342 g of sucrose, 20 g of agarpowder, 20 ml of COVE salt solution, and deionised water to 1000 ml. Themedium was sterilized by autoclaving at 15 psi for 15 minutes(Bacteriological Analytical Manual, 8th Edition, Revision A, 1998). Themedium was cooled to 60° C. and 10 mM acetamide, Triton X-100 (50 μl/500ml) was added.

COVE-N-Agar tubes were composed of 218 g Sorbitol, 10 g Dextrose, 2.02 gKNO₃, 25 g Agar, 50 ml Cove salt solution, and deionised water up to1000 ml.

COVE salt solution was composed of 26 g of MgSO₄.7H₂O, 26 g of KCL, 26 gof KH₂PO₄, 50 ml of COVE trace metal solution, and deionised water to1000 ml.

COVE trace metal solution was composed of 0.04 g of Na₂B₄O₇.10H₂O, 0.4 gof CuSO₄.5H₂O, 1.2 g of FeSO₄.7H₂O, 0.7 g of MnSO₄.H₂O, 0.8 g ofNa₂MoO₄.2H₂O, 10 g of ZnSO₄.7H₂O, and deionised water to 1000 ml.

Protease Assays Kinetic Suc-AAPF-pNA Assay:

-   pNA substrate: Suc-AAPF-pNA (Bachem L-1400).-   Temperature: Room temperature (25° C.)-   Assay buffers: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100    mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100 adjusted to    pH-values 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, and 11.0    with HCl or NaOH.

20 μl protease sample (diluted in 0.01% Triton X-100) was mixed with 100μl assay buffer. The assay was started by adding 100 μl pNA substrate(50 mg dissolved in 1.0 ml DMSO and further diluted 45× with 0.01%Triton X-100). The increase in OD₄₀₅ was monitored as a measure of theprotease activity.

Endpoint Suc-AAPF-pNA Assay:

-   pNA substrate: Suc-AAPF-pNA (Bachem L-1400).-   Temperature: controlled (assay temperature).-   Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100    mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100, pH 4.0

200 μl pNA substrate (50 mg dissolved in 1.0 ml DMSO and further diluted45× with the Assay buffer) were pipetted in an Eppendorf tube and placedon ice. 20 μl protease sample (diluted in 0.01% Triton X-100) was added.The assay was initiated by transferring the Eppendorf tube to anEppendorf thermomixer, which was set to the assay temperature. The tubewas incubated for 15 minutes on the Eppendorf thermomixer at its highestshaking rate (1400 rpm). The incubation was stopped by transferring thetube back to the ice bath and adding 600 μl 500 mM H₃BO₃/NaOH, pH 9.7.The tube was mixed and 200 μl mixture was transferred to a microtiterplate, which was read at OD₄₀₅. A buffer blind was included in the assay(instead of enzyme). OD₄₀₅(Sample)−OD₄₀₅(Blind) was a measure ofprotease activity.

Protazyme AK Assay:

-   Substrate: Protazyme AK tablet (cross-linked and dyed casein; from    Megazyme)-   Temperature: controlled (assay temperature).-   Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100    mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100, pH 6.5.

A Protazyme AK tablet was suspended in 2.0 ml 0.01% Triton X-100 bygentle stirring. 500 μl of this suspension and 500 μl assay buffer weredispensed in an Eppendorf tube and placed on ice. 20 μl protease sample(diluted in 0.01% Triton X-100) was added. The assay was initiated bytransferring the Eppendorf tube to an Eppendorf thermomixer, which wasset to the assay temperature. The tube was incubated for 15 minutes onthe Eppendorf thermomixer at its highest shaking rate (1400 rpm). Theincubation was stopped by transferring the tube back to the ice bath.Then the tube was centrifuged in an ice cold centrifuge for a fewminutes and 200 μl supernatant was transferred to a microtiter plate,which was read at OD₆₅₀. A buffer blind was included in the assay(instead of enzyme). OD₆₅₀(Sample)−OD₆₅₀(Blind) was a measure ofprotease activity.

Kinetic Suc-AAPX-pNA Assay:

-   pNA substrates: Suc-AAPA-pNA (Bachem L-1775)    -   Suc-AAPR-pNA (Bachem L-1720)    -   Suc-AAPD-pNA (Bachem L-1835)    -   Suc-AAPI-pNA (Bachem L-1790)    -   Suc-AAPM-pNA (Bachem L-1395)    -   Suc-AAPV-pNA (Bachem L-1770)    -   Suc-AAPL-pNA (Bachem L-1390)    -   Suc-AAPE-pNA (Bachem L-1710)    -   Suc-AAPK-pNA (Bachem L-1725)    -   Suc-AAPF-pNA (Bachem L-1400)-   Temperature: Room temperature (25° C.)-   Assay buffer: 100 mM succinic acid, 100 mM HEPES, 100 mM CHES, 100    mM CABS, 1 mM CaCl₂, 150 mM KCl, 0.01% Triton X-100, pH 4.0 or pH    9.0.

20 μl protease (diluted in 0.01% Triton X-100) was mixed with 100 μlassay buffer. The assay was started by adding 100 μl pNA substrate (50mg dissolved in 1.0 ml DMSO and further diluted 45× with 0.01% TritonX-100). The increase in OD₄₀₅ was monitored as a measure of the proteaseactivity.

o-Phthaldialdehyde (OPA) Assay:

This assay detects primary amines and hence cleavage of peptide bonds bya protease can be measured as the difference in absorbance between aprotease treated sample and a control sample. The assay is conductedessentially according to Nielsen et al. (Nielsen et al., 2001, Improvedmethod for determining food protein degree of hydrolysis, J. Food Sci.66: 642-646).

500 μl of sample is filtered through a 100 kDa Microcon centrifugalfilter (60 min, 11,000 rpm, 5° C.). The samples are dilutedappropriately (e.g., 10, 50 or 100 times) in deionizer water and 25 μlof each sample is loaded into a 96 well microtiter plate (5 replicates).200 μl OPA reagent (100 mM di-sodium tetraborate decahydrate, 3.5 mMsodium dodecyl sulphate (SDS), 5.7 mM di-thiothreitol (DDT), 6 mMo-phthaldialdehyde) is dispensed into all wells, the plate is shaken (10sec, 750 rpm) and absorbance measured at 340 nm.

Strain

The strain Meripilus giganteus was isolated from a fruiting bodycollected in Denmark in 1993 by Novozymes.

An in-house Trametes cf. versicolor strain identified by ITS (InternalTranscribed Spacer) sequencing was used as the source of the proteasegene SEQ ID NO: 15.

A second Trametes vesicolor strain sequenced by Genome Canada(www.fungalgenomics.ca) was used to identify the protease gene SEQ IDNO: 20.

Escherichia coli Top-10 strain purchased from Invitrogen (LifeTechnologies, Carlsbad, Calif., USA) was used to propagate theexpression vectors.

Aspergillus oryzae MT3568 strain was used for heterologous expression ofthe gene encoding polypeptides having homology with polypeptides withprotease activity. A. oryzae MT3568 is an amdS (acetamidase) disruptedgene derivative of Aspergillus oryzae JaL355 (WO 02/40694) in which pyrGauxotrophy was restored by disrupting the A. oryzae acetamidase (amdS)gene with the pyrG gene.

Example 1: Recombinant Expression of the S53 Protease 3 from Meripilusgiganteus (SEQ ID NO: 3)

In order to obtain material for testing and characterization of the S53Protease 3 from Meripilus giganteus, the DNA sequence from Seq ID NO: 1was cloned in an Aspergillus expression vector and expressed inAspergillus oryzae.

The S53 Protease 3 gene from Meripilus giganteus was sub-cloned into theAspergillus expression vector pMStr100 (WO 2010/009400) by amplifyingthe coding region without the stop codon of the DNA in Seq ID NO: 1 fromthe cDNA plasmid clone, pA2PR22, with standard PCR techniques using thefollowing primers:

597 (SEQ ID NO: 13) TAGGGATCCTCACGATGGTCGCCACCAGCT 598 (SEQ ID NO: 14)CAGGCCGACCGCGGTGAG

The PCR product was restricted with BamHI and ligated into the BamHI andNruI sites of pMStr100, resulting in an in-frame fusion with theC-terminal tag sequence RHQHQHQH (stop) in the expression vector. TheS53 Protease 3 gene in the resulting Aspergillus expression construct,pMStr121, was sequenced, and the protease coding portion of the sequencewas confirmed to agree with the original coding sequence of SEQ IDNO: 1. The in-frame fusion to the tag encoding sequence was alsoconfirmed, resulting in the sequence in SEQ ID NO: 3, which encodes thepeptide sequence in SEQ ID NO: 4.

The Aspergillus oryzae strain BECh2 (WO 00/39322) was transformed withpMStr121 using standard techniques as described by Christensen et al.,1988, Biotechnology 6: 1419-1422 and WO 2004/032648. To identifytransformants producing the recombinant protease, the transformants andBECh2 were cultured in 10 ml of YP+2% glucose medium at 30° C. and 200rpm. Samples were taken after 3 days growth and resolved with SDS-PAGEto identify recombinant protease production. A novel band between 35 and50 kDa was observed in cultures of transformants that was not observedin cultures of the untransformed BECh2. Several transformants thatappeared to express the recombinant protease at high levels were furthercultured in 100 ml of YP+2% glucose medium in 500 ml shake flasks at 30°C. and 200 rpm. Samples were taken after 2, 3, and 4 days growth andexpression levels compared by resolving the samples with SDS-PAGE. Asingle transformant that expressed the recombinant protease atrelatively high levels was selected and designated EXP01737. EXP01737was isolated twice by dilution streaking conidia on selective mediumcontaining 0.01% TRITON® X-100 to limit colony size and fermented inYP+2% glucose medium in shake flasks as described above to providematerial for purification. The shake flask cultures were harvested after4 days growth and fungal mycelia was removed by filtering thefermentation broth through Miracloth (Calbiochem) then purified asdescribed in example 2.

YP+2% Glucose Medium

10 g yeast extract 20 g peptone water to 1 L autoclave at 121° C., 20minutes add 100 ml 20% sterile glucose solution

Example 2: Purification of the S53 Protease 3 from Meripilus giganteuswith C-Terminal HQ-Tag

The culture broth was centrifuged (20000×g, 20 min) and the supernatantwas carefully decanted from the precipitate. The supernatant wasfiltered through a Nalgene 0.2 μm filtration unit in order to remove therest of the Aspergillus host cells. The 0.2 μm filtrate was transferredto 10 mM Succinic acid/NaOH, pH 3.5 on a G25 Sephadex column (from GEHealthcare). The G25 sephadex transferred enzyme was applied to aQ-sepharose FF column (from GE Healthcare) equilibrated in 10 mMSuccinic acid/NaOH, pH 3.5. The run-through and wash with 10 mM Succinicacid/NaOH, pH 3.5 was collected and contained the S53 protease (activityconfirmed using the Kinetic Suc-AAPF-pNA assay at pH 4). The pH of therun-through and wash fraction was adjusted to pH 3.25 with 1 M HCl whilemixing the fraction thoroughly. The pH-adjusted solution was applied toa SP-sepharose FF column (from GE Healthcare) equilibrated in 10 mMSuccinic acid/NaOH, pH 3.25. After washing the column extensively withthe equilibration buffer, the protease was eluted with a linear NaClgradient (0-->0.5 M) in the same buffer over ten column volumes.Fractions from the column were analysed for protease activity (using theKinetic Suc-AAPF-pNA assay at pH 4) and peak-fractions were pooled.Solid ammonium sulphate was added to the pool to 2.0 M final (NH₄)₂SO₄concentration. The enzyme solution was applied to a Phenyl-Toyopearlcolumn (from TosoHaas) equilibrated in 10 mM Succinic acid/NaOH, 2.0 M(NH₄)₂SO₄, pH 3.25. After washing the column extensively with theequilibration buffer, the S53 protease was eluted with a linear gradientbetween the equilibration buffer and 10 mM Succinic acid/NaOH, pH 3.25over ten column volumes. Fractions from the column were analyzed forprotease activity (using the Kinetic Suc-AAPF-pNA assay at pH 4).Fractions with high activity were pooled and transferred to 10 mMSuccinic acid/NaOH, pH 3.5 on a G25 sephadex column (from GEHealthcare). The G25 sephadex transferred protease was applied to aSP-sepharose HP column (from GE Healthcare) equilibrated in 10 mMSuccinic acid/NaOH, pH 3.5. After washing the column extensively withthe equilibration buffer, the protease was eluted with a linear NaClgradient (0-->0.5 M) in the same buffer over five column volumes.Fractions constituting the major peak from the column were pooled as thepurified product. The purified product was analysed by SDS-PAGE and onemajor band was seen on the gel and three minor bands. EDMAN N-terminalsequencing of the bands showed that all the bands were related to theS53 protease and therefore we expect that the minor bands representsnicking of some of the S53 protease molecules. The purified product wasused for further characterization.

Example 3: Characterization of the S53 Protease 3 from Meripilusgiganteus with C-Terminal HQ-Tag

The Kinetic Suc-AAPF-pNA assay was used for obtaining the pH-activityprofile and the pH-stability profile (residual activity after 2 hours atindicated pH-values). For the pH-stability profile the protease wasdiluted 10× in the different assay buffers to reach the pH-values ofthese buffers and then incubated for 2 hours at 37° C. After incubation,the pH of the protease incubations was transferred to the same pH-value,before assay for residual activity, by dilution in the pH 4.0 Assaybuffer. The Endpoint Suc-AAPF-pNA assay was used for obtaining thetemperature-activity profile at pH 4.0. The Kinetic Suc-AAPX-pNA assayand ten different Suc-AAPX-pNA substrates were used for obtaining theP1-specificity of the enzyme at pH 4.0.

The results are shown in tables 2-5 below. Data for Protease 10R areincluded in the tables. For table 2, the activities are relative to theoptimal pH for the enzymes. For table 3, the activities are residualactivities relative to samples, which were kept at stable conditions (5°C., pH 4.0 for the S53 protease 3 from Meripilus giganteus (from example2); 5° C., pH 9.0 for Protease 10R). For table 4, the activities arerelative to the optimal temperature for the enzyme (pH 4.0 for the S53protease 3 from Meripilus giganteus (from example 2); pH 6.5 forProtease 10R). For table 5, the activities are relative to the bestsubstrate for the enzymes (Suc-AAPL-pNA for the S53 protease 3 fromMeripilus giganteus (from example 2)). The Protazyme AK assay was usedfor obtaining the temperature-activity profile at pH 6.5 for Protease10R.

The pH-activity on the Suc-AAPF-pNA substrate, the pH-stability profile(residual activity after 2 hours at 37° C.), the temperature activityprofile on Suc-AAPF-pNA at pH 4.0 and the P1-specificity on 10Suc-AAPF-pNA substrates at pH 4.0 for the S53 protease 3 from Meripilusgiganteus (from example 2) compared with the data for Protease 10R arealso shown in FIGS. 1-4. For Protease 10R, the temperature activityprofile is on Protazyme AK at pH 6.5 and the P1-specificity is at pH9.0.

TABLE 2 pH-activity profile at 25° C. as determined using the kineticSuc-AAPF-pNA assay S53 protease 3 from Meripilus giganteus pH (fromexample 2) Protease 10R 2 0.38 — 3 0.95 0.00 4 1.00 0.02 5 0.27 0.07 60.02 0.21 7 0.00 0.44 8 0.00 0.67 9 0.00 0.88 10 0.00 1.00 11 0.00 0.93

TABLE 3 pH-stability profile (residual activity after 2 hours at 37° C.)as determined using the kinetic Suc-AAPF-pNA assay S53 protease 3 fromMeripilus giganteus pH (from example 2) Protease 10R 2 0.01 0.78 3 0.991.03 4 0.96 0.99 5 0.94 1.00 6 0.87 1.03 7 0.69 1.01 8 0.01 0.98 9 0.010.99 10 0.01 0.99 11 0.01 0.86 After 2 hours at 1.00 1.00 5° C. (at pH4) (at pH 9)

TABLE 4 Temperature activity profile at pH 4.0 or pH 6.5 as determinedusing the endpoint Suc-AAPF-pNA assay S53 protease 3 from Meripilusgiganteus Protease 10R Temp (° C.) (from example 2, pH 4) (pH 6.5) 150.07 0.01 25 0.23 0.02 37 0.58 0.06 50 1.00 0.13 60 0.44 0.35 70 0.080.96 80 — 1.00 90 — 0.18

TABLE 5 P1-specificity on 10 Suc-AAPX-pNA substrates at pH 4.0 or pH 9.0at 37° C. as determined using the kinetic Suc-AAPX-pNA assay S53protease 3 from Meripilus giganteus Protease 10R Suc-AAPX-pNA (fromexample 2, pH 4) (pH 9) Suc-AAPA-pNA 0.01 0.13 Suc-AAPR-pNA 0.00 0.09Suc-AAPD-pNA 0.06 0.00 Suc-AAPI-pNA 0.00 0.00 Suc-AAPM-pNA 0.53 0.78Suc-AAPV-pNA 0.00 0.01 Suc-AAPL-pNA 1.00 0.18 Suc-AAPE-pNA 0.05 0.00Suc-AAPK-pNA 0.00 0.08 Suc-AAPF-pNA 0.99 1.00Other Characteristics for the S53 Protease 3 from Meripilus giganteus(from Example 2)

Determination of the N-terminal sequence was: AIPASCASTI.

The relative molecular weight as determined by SDS-PAGE was approx. M,=43 kDa.

Confirmation of C-Terminal HQ-Tag Attached to Mature Sequence

This sample was buffer exchanged with 50 mM sodium acetate buffer pH 5.5using a Vivaspin ultrafiltration unit fitted with a 10 kDa cut offfilter. Following buffer exchange, 2 μL of endoglycosidase H was addedand the sample was then incubated at 5° C. overnight. Note: For everydeglycosylated N-linked site one N-acetyl hexosamine residue remains onthe protein backbone increasing the molecular weight with 203.19 Da persite. The sample was then analyzed by mass-spectrometry.

The molecular weight determined by intact molecular weight analysis ofthe major peak was: 38088.6 Da, corresponding to within 1.8 Da of themature sequence plus-RHQHQ plus a single acetyl hexosamine and onenon-crosslinked cysteine residue.

The molecular weight determined by intact molecular weight analysis ofthe secondary peak was: 37961 Da, corresponding to within 2.3 Da of themature sequence plus-RHQH plus a single acetyl hexosamine and onenon-crosslinked cysteine residue.

The mature sequence (from EDMAN N-terminal sequencing data and intactmolecular weight analysis):

(SEQ ID NO: 6) AIPASCASTITPACLQAIYGIPTTKATQSSNKLAVSGFIDQFANKADLKSFLAQFRKDISSSTTFSLQTLDGGENDQSPSEAGIEANLDIQYTVGLATGVPTTFISVGDDFQDGNLEGFLDIINFLLGESNPPQVLTTSYGQNENTISAKLANQLCNAYAQLGARGTSILFASGDGGVSGSQSAHCSNFVPTFPSGCPFMTSVGATQGVSPETAAAFSSGGFSNVFGIPSYQASAVSGYLSALGSTNSGKFNRSGRGFPDVSTQGVDFQIVSGGQTIGVDGTSCASPTFASVISLVNDRLIAAGKSPLGFLNPFLYSSAGKAALNDVTSGSNPGCSTNGFPAKAGWDPVTGLGTPNFAKLLTAVGLRHQHQ.

The calculated molecular weight from this mature sequence is 37882.6 Da.

Example 4: Soybean-Maize Meal Activity Assay

An end-point assay using soybean-maize meal as substrate was used forobtaining the activity profile of the proteases at pH 3-7.

-   Substrate: Soybean meal-maize meal mixed in a 30:70 ratio.-   Assay buffers: 9 buffers containing 100 mM succinic acid, 100 mM    HEPES, 100 mM CHES, 100 mM CAPS, 1 mM CaCl₂, 150 mM KCl, 0.01%    Triton X-100 were prepared and adjusted using HCl or NaOH to a pH    value such that after soybean-maize meal substrate (1 g) had been    mixed with assay buffer (10 mL) to give a slurry, the final pH of    the slurry was one of the following pH's: 3.0, 4.0, 5.0, 6.0, 7.0,    8.0, 9.0, 10.0 and 11.0.

Substrate slurry (2 mL) was mixed for 30 min before protease additionand incubated for 3 hours at 40° C. (500 rpm). Protease (200 mg enzymeprotein/kg dry matter) was dissolved in 100 μl 100 mM sodium acetatebuffer (9.565 g/L NaOAc, 1.75 g/L acetic acid, 5 mM CaCl₂, 0.01% BSA,0.01% Tween20, pH 6.0) and added. Samples were centrifuged (10 min, 4000rpm, 0° C.) and the supernatants collected for analysis using theo-Phthaldialdehyde (OPA) assay.

The results are shown in Table 6 below. The proteolytic activity of theS53 protease 3 from Meripilus giganteus (from example 2) onsoybean-maize meal is at its highest at pH 3 and decreases withincreasing pH. At pH 5, the S53 protease 3 from Meripilus giganteus(from example 2) is as active on soybean-maize meal as Protease 10R,whereas at pH 3 and pH 4 the S53 protease 3 from Meripilus giganteus(from example 2) is much more active than Protease 10R. These resultsindicate that the S53 protease 3 from Meripilus giganteus (from example2) could be efficient for obtaining protein hydrolysis in the uppergastro-intestinal tract of monogastric animals such as, e.g., pigs andpoultry leading to improved utilization of feed protein in thesespecies.

TABLE 6 Protease activity (OD340 × dilution factor) on soybean-maizemeal at pH 3.0, 4.0, 5.0, 6.0 and 7.0 S53 protease 3 from MeripilusProtease 10R giganteus (from example 2) Standard pH Average Standarddeviation Average deviation 3.0 3.02 0.08 0.22 0.06 4.0 1.31 0.06 0.300.10 5.0 0.64 0.03 0.71 0.01 6.0 0.19 0.04 1.81 0.14 7.0 0.02 0.04 2.920.11

FIG. 5 shows the activity (OD₃₄₀× dilution factor) on soybean-maize mealof the S53 protease 3 from Meripilus giganteus (from example 2) comparedto the 10R protease.

Example 5: Proteolytic Activity on Crop, Gizzard and Ileum Digesta fromBroiler Chickens

Crop, gizzard and ileum digesta material from 21 day old broilerchickens fed a corn-soy diet was collected; freeze dried and groundusing a small coffee mill. The ground samples were suspended (47% w/v)in the following buffers and left to hydrate at 4° C. overnight (nostirring):

-   Crop buffer: 100 mM HEPES, 1 mM CaCl₂.2H₂O, 150 mM KCl, 0.01% Triton    X-100, adjusted to pH 5 using HCl-   Gizzard buffer: 100 mM succinic acid, 1 mM CaCl₂.2H₂O, 150 mM KCl,    0.01% Triton X-100, adjusted to pH 1.67 using HCl-   Ileum buffer: 100 mM HEPES, 1 mM CaCl₂.2H₂O, 150 mM KCl, 0.01%    Triton X-100, adjusted to pH 7.2 using HCl

The resulting pH was: pH 5 in crop samples; pH 3 in gizzard samples; andpH 7 in ileum samples. The suspensions were heated to 40° C. and 1 mlwas dispensed into tubes kept at 40° C. Three tubes representing blank(T₀) were immediately centrifuged (3000×g, 0° C., 10 min) and thesupernatants frozen. Either enzyme (200 mg enzyme protein/kg substrate)in 50 μL 100 mM sodium acetate buffer (9.565 g/I NaOAc, 1.75 g/I aceticacid, 5 mM CaCl₂, 0.01% BSA, 0.01% Tween20, pH 6.0) or just sodiumacetate buffer (50 μL) for the blank samples was added to the tubes andcrop and ileum samples were incubated for 3 hours (T₃) while the gizzardsamples were incubated for 1 hour (T₁) at 40° C. while shaking (500rpm). The samples were centrifuged (3000×g, 0° C., 10 min) andsupernatants recovered and frozen. The proteolytic activity wasdetermined by analyzing primary amines using the o-phthaldialdehyde(OPA) assay.

The results are shown in Table 7. For each of the digesta types (crop,gizzard and ileum) there was a significant difference between the levelof primary amines in the blank T₀ sample and the blank samples incubatedfor 1 or 3 hours. This difference can be ascribed to activity ofproteases present in the substrate and originating from either the dietraw materials or the animal. During incubation of the crop and gizzarddigesta the S53 protease 3 from Meripilus giganteus (from example 2)further increased the level of primary amines compared to the blanksample, demonstrating that the protease had a proteolytic activity onthis substrate under the given conditions. The S53 protease 3 fromMeripilus giganteus (from example 2) performed significantly betterduring crop and gizzard incubation than Protease 10R, indicating thatthe S53 protease 3 from Meripilus giganteus (from example 2) could bemore efficient for feed protein hydrolysis in the uppergastro-intestinal tract of poultry leading to improved proteindigestibility. As expected the S53 protease 3 from Meripilus giganteus(from example 2) did not significantly increase the level of free aminesduring ileum incubation at pH 7.

TABLE 7 Proteolytic activity of the S53 protease 3 from Meripilusgiganteus (from example 2) compared to Protease 10R when incubated withbroiler digesta and expressed as level of primary amines measured by theOPA assay (OD₃₄₀ × dilution factor) Treatment Crop (3 hours) Gizzard (1hour) Ileum (3 hours) Blank (T₀) 2.21 ± 0.02^(d) 2.95 ± 0.02^(c)  9.37 ±0.08^(c) Blank 3.54 ± 0.02^(c) 3.94 ± 0.08^(b) 14.40 ± 0.66^(ab) S53protease 3 from 4.13 ± 0.03^(a) 4.37 ± 0.05^(a) 14.20 ± 0.19^(ab)Meripilus giganteus (from example 2) Protease 10R 3.85 ± 0.07^(b) 3.87 ±0.21^(b) 14.74 ± 0.15^(a) ^(a,b,c)Values within a column that are notconnected by the same superscript letters are statistically different asdetermined by the Tukey Kramer test (α = 0.05) provided by the ANOVAprocedure (SAS Institute Inc.).

FIG. 6 shows the level of free amines (OD₃₄₀× dilution factor) in blankT₀ samples, blank samples and samples incubated with the S53 protease 3from Meripilus giganteus (from example 2) or the 10R protease. Thesubstrate for the incubation was digesta material from the crop, gizzardor ileum of broiler chickens.

Example 6: Thermostability

An aliquot of the protein sample of protease (purified as described inExample 2 or 12) is either desalted or buffer-changed into 20 mMNa-acetate, pH 4.0 using a prepacked PD-10 column or dialysed against2×500 ml 20 mM Na-acetate, pH 4.0 at 4° C. in a 2-3 h step followed byan overnight step. The sample is 0.45 μm filtered and diluted withbuffer to approx. 2 A280 units. The dialysis buffer is used as referencein Differential Scanning calorimetry (DSC). The samples are degassedusing vacuum suction and stirring for approx. 10 minutes.

A DSC scan is performed on a MicroCal VP-DSC at a constant scan rate of1.5° C./min from 20-90° C. Data-handling is performed using the MicroCalOrigin software (version 4.10), and the denaturation temperature, T_(d)(also called the melting temperature, T_(m)) is defined as thetemperature at the apex of the peak in the thermogram.

Example 7: Steam Stability

Residual activity of the protease after steam treatment may be evaluatedusing the following assay.

In these experiments, a modified set-up is used whereby the steam isprovided from a steam generator and led into the box. The samples placedon a plate are inserted into the box through a drawer when thetemperature has reached ca. 93-94° C. Upon the insertion of the samplesthe temperature drops 4° C. Incubation is performed for 30 seconds whilethe temperature remains approximately constant at 90° C. Thereafter theplate is quickly removed from the box, the samples placed on ice,re-suspended and evaluated with respect to protease activity using,e.g., the Suc-AAPF-pNA or o-Phthaldialdehyde (OPA) assay. Each enzymesample is compared to a similar sample that had not been steam treatedin order to calculate residual activity.

Example 8: Pelleting Stability Tests

The enzyme granulation is performed in a manner as described in U.S.Pat. No. 4,106,991, Example 1. The obtained granulate is dried in afluid bed to a water content below 1% and sifted to obtain a productwith the particle range 250 μm to 850 μm. Finally, the product is coatedwith palm oil and calcium carbonate in a manner as described in U.S.Pat. No. 4,106,991, Example 22.

Approximately 50 g enzyme granulate is pre-mixed with 10 kg feed for 10minutes in a small horizontal mixer. This premix is mixed with 90 kgfeed for 10 minutes in a larger horizontal mixer. From the mixer, thefeed is led to the conditioner (a cascade mixer with steam injection) ata rate of approximately 300 kg/hour. The conditioner heats up the feedto 95° C. (measured at the outlet) by injecting steam. The residencetime in the conditioner is 30 seconds. From the conditioner, the feed isled to a Simon Heesen press equipped with 3.0×35 mm horizontal die andpressed to pellets with a length of around 15 mm. After the press, thepellets are placed in an air cooler and cooled for 15 minutes.

The protease activity is measured using the Suc-AAPF-pNA assay prior topelleting and in the feed pellets after pelleting. Pelleting stabilityis determined by comparing the protease activity in pelleted feedrelative to the activity in non-pelleted feed.

Example 9: Cloning of Two Protease Genes from Trametes cf. Versicolorand Trametes versicolor

Based on the gene sequences identified, SEQ ID NO: 15 from a Trametescf. versicolor strain (see strain section) and SEQ ID NO: 20 from aTrametes versicolor strain (see strain section) two synthetic coding DNAsequences (CDS) with codon optimization for Aspergillus oryzaeexpression were designed (SEQ ID NO: 17 and SEQ ID NO: 22,respectively). Those two CDS sequences were synthesized by GeneArt®(Life Technologies, Carlsbad, Calif., USA) in a pMA-T vector at a 5 μgscale with two flanking sites BamHI in 5′ and HindIII in 3′ compatiblewith the expression vector pDAu109 (WO 2005/042735). 1 μg of thoseplasmids was subsequently digested with the restriction enzymes BamHIand HindIII from NEB (New England Biolabs, Frankfurt am Main Germany)following manufacturer's recommendations, and the resulting fragmentswere separated by 1% agarose gel electrophoresis using TAE buffer. The1.7 kb fragment corresponding to the synthetic protease genes wereexcised from the gel and purified using a GFX® PCR DNA and Gel BandPurification Kit (GE Healthcare, HiHerød, Denmark) following themanufacturer's instructions. 100 ng of those inserts were cloned in theexpression vector pDAu109 (WO 2005/042735) previously digested withBamHI and HindIII by ligation with a T4 ligase from NEB (New EnglandBiolabs, Frankfurt am Main Germany) following the manufacturer'sinstructions.

A 2.5 μl volume of the diluted ligation mixture was used to transform E.coli TOP10 chemically competent cells (Life Technologies, Carlsbad,Calif., USA). Three colonies were selected from LB agar platescontaining 100 μg of ampicillin per ml for each construct and cultivatedovernight in 3 ml of LB medium supplemented with 100 μg of ampicillinper ml. Plasmid DNA was purified using a Qiagen Spin Miniprep kit (Cat.27106) (QIAGEN GmbH, Hilden, Germany) according to the manufacturer'sinstructions. The Trametes cf. versicolor and Trametesversicolorprotease synthetic sequences were verified by Sangersequencing before heterologous expression. The plasmids designated asMDQM0673-1 and MDQM0584-1 (holding the CDS SEQ ID NO: 17 and SEQ ID NO:22, respectively) were selected for protoplast transformation andheterologous expression of its encoded proteases in an Aspergillusoryzae host cell MT3568 (described in the strain chapter).

Example 10: Transformation of Aspergillus oryzae with the Gene EncodingProteases from Trametes cf. Versicolor and Trametes versicolor andSelection of the Best Transformants

Protoplasts of Aspergillus oryzae MT3568 (see strains chapter) wereprepared according to WO 95/02043. One hundred μl of protoplasts weremixed with 2.5-10 μg of either of the Aspergillus expression vectorsMDQM0673-1 and MDQM0584-1 (Example 9), 250 μl of 60% PEG 4000(Applichem, Darmstadt, Germany) (polyethylene glycol, molecular weight4,000), 10 mM CaCl₂, and 10 mM Tris-HCl pH 7.5 and gently mixed. Themixture was incubated at 37° C. for 30 minutes and the protoplasts werespread onto COVE plates for selection. After incubation for 4-7 days at37° C. spores of eight transformants were inoculated into 0.5 ml ofDAP4C-1 medium supplemented lactic acid and with diammonium phosphate in96 deep well plates. After 4 days cultivation at 30° C., the culturebroths were analyzed by SDS-PAGE using Novex® 4-20% Tris-Glycine Gel(Invitrogen Corporation, Carlsbad, Calif., USA) to identify thetransformants producing the largest amount of recombinant protease fromTrametes versicolor.

Based on the band intensity of the SDS-PAGE gel, spores of the besttransformant were spread on COVE-Sucrose-T plates containing 0.01%TRITON® X-100 in order to isolate single colonies. The spreading wasrepeated twice in total on COVE-Sucrose-T plates, and then a singlecolony was spread on a COVE-N-Agar tube until sporulation.

Example 11: Fermentation of Aspergillus oryzae Transformed with the GeneEncoding Proteases from Trametes cf. Versicolor and Trametes versicolor

150 ml of DAP4C-1 medium supplemented with 5 ml of 20% lactic acid and3.5 ml of 50% diammonium phosphate and spores from the besttransformants were cultivated in shake flasks during 4 days at atemperature of 30° C. under 100 rpm agitation. Culture broths wereharvested by filtration using a 0.2 μm filter device and used forfurther characterization.

Example 12: Purification of the S53 Protease 1 from Trametes cfVersicolor

The culture broth was centrifuged (20000×g, 20 min) and the supernatantwas carefully decanted from the precipitate. The supernatant wasfiltered through a Nalgene 0.2 μm filtration unit in order to remove therest of the Aspergillus host cells. The 0.2 μm filtrate was transferredto 10 mM Succinic acid/NaOH, pH 3.5 on a G25 Sephadex column (from GEHealthcare). The G25 sephadex transferred enzyme was applied to aSP-sepharose FF column (from GE Healthcare) equilibrated in 10 mMSuccinic acid/NaOH, pH 3.5. After washing the column extensively withthe equilibration buffer, the protease was eluted with a linear NaClgradient (0-->1.0 M) in the same buffer over ten column volumes.Fractions from the column were analysed for protease activity (using theKinetic Suc-AAPF-pNA assay at pH 4) and peak-fractions were analysed bySDS-PAGE. Fractions with one band only on the coomassie stained SDS-PAGEgel were pooled as the purified product. The purified product was usedfor further characterization.

Example 13: Characterization of the S53 Protease 1 from Trametes cfVersicolor (SEQ ID NO: 19)

The Kinetic Suc-AAPF-pNA assay was used for obtaining the pH-activityprofile. The Endpoint Suc-AAPF-pNA assay was used for obtaining thepH-stability profile (residual activity after 2 hours at indicatedpH-values) and the temperature-activity profile at pH 4.0. For thepH-stability profile the protease was diluted 7× in the different Assaybuffers to reach the pH-values of these buffers and then incubated for 2hours at 37° C. After incubation, the pH of the protease incubations wastransferred to the same pH-value, before assay for residual activity, bydilution in the pH 4.0 Assay buffer. The Kinetic Suc-AAPX-pNA assay andten different Suc-AAPX-pNA substrates were used for obtaining theP1-specificity of the enzyme at pH 4.0.

The results are shown in Tables 8-11 below. Data for S53 protease 3 fromMeripilus giganteus (from example 2) and protease 10R are included inthe tables. For Table 8, the activities are relative to the optimal pHfor the enzymes. For Table 9, the activities are residual activitiesrelative to samples, which were kept at stable conditions (5° C., pH4.0). For Table 10, the activities are relative to the optimaltemperature at pH 4.0 for the enzyme. For Table 11, the activities arerelative to the best substrate for the enzymes (Suc-AAPL-pNA for the S53protease 1 from Trametes cf versicolor).

TABLE 8 pH-activity profile at 25° C. as determined using the kineticSuc-AAPF-pNA assay S53 protease 1 from S53 protease 3 from Trametes cfversicolor Meripilus giganteus pH (from example 12) (from example 2)Protease 10R 2 0.00 0.38 — 3 0.75 0.95 0.00 4 1.00 1.00 0.02 5 0.32 0.270.07 6 0.02 0.02 0.21 7 0.00 0.00 0.44 8 0.00 0.00 0.67 9 0.00 0.00 0.8810 0.00 0.00 1.00 11 0.00 0.00 0.93

TABLE 9 pH-stability profile (residual activity after 2 hours at 37° C.)as determined using the kinetic Suc-AAPF-pNA assay S53 protease 1 fromS53 protease 3 from Trametes cf versicolor Meripilus giganteus pH (fromexample 12) (from example 2) Protease 10R 2 0.01 0.01 0.78 3 0.31 0.991.03 4 0.94 0.96 0.99 5 0.92 0.94 1.00 6 0.10 0.87 1.03 7 0.03 0.69 1.018 0.01 0.01 0.98 9 0.01 0.01 0.99 10 0.01 0.01 0.99 11 0.00 0.01 0.86After 2 1.00 1.00 1.00 hours at (at pH 4) (at pH 4) (at pH 9) 5° C.

TABLE 10 Temperature activity profile at pH 4.0 or pH 6.5 as determinedusing the endpoint Suc-AAPF-pNA assay S53 protease 1 from S53 protease 3from Temp Trametes cf versicolor Meripilus giganteus Protease 10R (° C.)(from example 12, pH 4) (from example 2, pH 4) (pH 6.5) 15 0.16 0.070.01 25 0.36 0.23 0.02 37 1.00 0.58 0.06 50 0.79 1.00 0.13 60 0.16 0.440.35 70 0.08 0.08 0.96 80 — — 1.00 90 — — 0.18

TABLE 11 P1-specificity on 10 Suc-AAPX-pNA substrates at pH 4.0 or pH9.0 at 37° C. as determined using the kinetic Suc-AAPX-pNA assay S53protease 1 from S53 protease 3 from Trametes cf versicolor Meripilusgiganteus Protease (from (from 10R Suc-AAPX-pNA example 12, pH 4)example 2, pH 4) (pH 9) Suc-AAPA-pNA 0.01 0.01 0.13 Suc-AAPR-pNA 0.000.00 0.09 Suc-AAPD-pNA 0.04 0.06 0.00 Suc-AAPI-pNA 0.00 0.00 0.00Suc-AAPM-pNA 0.46 0.53 0.78 Suc-AAPV-pNA 0.00 0.00 0.01 Suc-AAPL-pNA1.00 1.00 0.18 Suc-AAPE-pNA 0.03 0.05 0.00 Suc-AAPK-pNA 0.00 0.00 0.08Suc-AAPF-pNA 0.81 0.99 1.00

The pH-activity on the Suc-AAPF-pNA substrate, the pH-stability profile(residual activity after 2 hours at 37° C.), the temperature activityprofile on Suc-AAPF-pNA at pH 4.0 and the P1-specificity on 10Suc-AAPF-pNA substrates at pH 4.0 for the S53 protease 1 from Trametescf versicolor compared with the data for the S53 protease 3 fromMeripilus giganteus are also shown as FIGS. 1-4 below.

Other Characteristics for the S53 Protease 1 from Trametes cf Versicolor

Determination of the N-terminal sequence was: AIPASCASTI.

The relative molecular weight as determined by SDS-PAGE was approx. M,=42 kDa.

Confirmation of the Mature Sequence for the S53 Protease 1 from Trametescf Versicolor

The purified sample was buffer exchanged with 50 mM sodium acetatebuffer pH 5.5 using a Vivaspin ultrafiltration unit fitted with a 10 kDacut off filter. Following buffer exchange, Endoglycosidase H was addedand the sample was incubated at 30° C. for 3 hours. Note: For eachdeglycosylated N-linked site one N-acetyl hexosamine residue remains onthe protein backbone increasing the molecular weight with 203.19 Da persite. The sample was then analyzed by mass-spectrometry.

The molecular weight determined by intact molecular weight analysis ofthe major peak was: 37467.6 Da, corresponding to within 0.41 Da of themature sequence plus a single acetyl hexosamine and one non-crosslinkedcysteine residue.

The mature sequence (from EDMAN N-terminal sequencing data and Intact MSdata):

(SEQ ID NO: 19) AVPASCASTITPACLQALYGIPTTKATQSSNKLAVSGFIDQFANSADLKTFLGKFRTDISSSTTFTLQTLDGGSNSQSSSQAGVEANLDIQYTVGLASAVPTIFISVGDDFQDGDLEGFLDIINFLLNESAPPQVLTTSYGQNENTISAKLANQLCNAYAQLGARGTSILFASGDGGVSGSQSSSCSKFVPTFPSGCPFMTSVGATQGINPETAADFSSGGFSNVFARPSYQSTAVSSYLTALGSTNSGKFNTSGRAFPDIATQGVDFEIVVSGRTEGVDGTSCASPTLAAIISLLNDRLIAAGKSPLGFLNPFLYSAAGTAALTDITSGSNPGCNTNGFPAKAGWDPVT GLGTPNFAKLLTAVGL.

The calculated molecular weight from this mature sequence is 37263.0 Da.

1-25. (canceled)
 26. A variant of the polypeptide of SEQ ID NO: 5, SEQID NO: 6, SEQ ID NO: 19 or SEQ ID NO: 24, or the mature polypeptide ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 21or SEQ ID NO: 23, comprising a substitution, deletion, and/or insertionat one or more positions, wherein the variant has protease activity andis at least 90% identical to the polypeptide of SEQ ID NO:
 5. 27. Thevariant of claim 26, which is at least 95% identical to the polypeptideof SEQ ID NO:
 5. 28. The variant of claim 26, which is at least 97%identical to the polypeptide of SEQ ID NO:
 5. 29. The variant of claim26, which is at least 98% identical to the polypeptide of SEQ ID NO: 5.30. The variant of claim 26, which is at least 99% identical to thepolypeptide of SEQ ID NO:
 5. 31. An animal feed additive comprising atleast one variant of claim 26; and at least one fat soluble vitamin,and/or at least one water soluble vitamin, and/or at least one tracemineral.
 32. The animal feed additive of claim 31, which furthercomprises of one or more amylases; galactanases; alpha-galactosidases;beta-glucanases; phospholipases; phytases; proteases; xylanases; or anymixture thereof.
 33. An animal feed having a crude protein content of 50to 800 g/kg and comprising at least one variant of claim
 26. 34. Amethod for improving the nutritional value of a protein, comprisingadding a variant of claim 26 to a composition comprising at least oneprotein or protein source.
 35. The method of claim 34, wherein theprotein source comprises soybean or soybean meal.
 36. A method forimproving the nutritional value of an animal feed, comprising adding avariant of claim 26 to the animal feed.
 37. An isolated polynucleotideencoding the variant of claim
 26. 38. A nucleic acid construct orexpression vector comprising the polynucleotide of claim 37 operablylinked to one or more control sequences that direct the production ofthe polypeptide in an expression host cell.
 39. A recombinant host cellcomprising the polynucleotide of claim 37 operably linked to one or morecontrol sequences that direct the production of the polypeptide.
 40. Thehost cell of claim 39, wherein the host is a filamentous fungus or ayeast.
 41. The host cell of claim 40, wherein the host is anAspergillus.
 42. The host cell of claim 39, wherein the host is aBacillus.
 43. A method of producing a variant having protease activity,comprising: (a) cultivating a host cell of claim 39 under conditionsconducive for production of the polypeptide; and (b) recovering thepolypeptide.